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Hormonal Regulation of Farm Animal Growth - K. Hossner (CABI, 2005) WW

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HORMONAL REGULATION
OF FARM ANIMAL GROWTH
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HORMONAL REGULATION
OF FARM ANIMAL GROWTH
K.L. Hossner
Department of Animal Sciences
Colorado State University
Fort Collins, Colorado
USA
CABI Publishing
CABI Publishing is a division of CAB International
CABI Publishing
CAB International
Wallingford
Oxfordshire OX10 8DE
UK
Tel: +44 (0)1491 832111
Fax: +44 (0)1491 833508
E-mail: [email protected]
Website: www.cabi-publishing.org
CABI Publishing
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USA
Tel: +1 617 395 4056
Fax: +1 617 354 6875
E-mail: [email protected]
© K.L. Hossner 2005. All rights reserved. No part of this publication may be
reproduced in any form or by any means, electronically, mechanically, by photocopying, recording or otherwise, without the prior permission of the
copyright owners. All queries to be referred to the publisher.
A catalogue record for this book is available from the British Library,
London, UK.
Library of Congress Cataloging-in-Publication Data
Hossner, K. L. (Kim L.)
Hormonal regulation of farm animal growth / K.L. Hossner.
p. cm.
Includes bibliographical references and index.
ISBN-10: 0-85199-080-0 (alk. paper)
ISBN-13: 978-0-85199-080-4 (alk. paper)
1. Livestock--Growth--Regulation. 2. Hormones. 3. Growth regulators. I. Title.
SF768.H67 2005
636.08’52--dc22
2005005787
ISBN 0 85199 080 0
Typeset by SPI Publisher Services, Pondicherry, India
Printed and bound in the UK by Cromwell Press, Trowbridge
Contents
1
Whole Animal Growth
Measuring Animal Growth
Embryonic and Fetal Growth
Growth Curves
Genetics, Nutrition and Environmental Effects
on Whole Animal Growth
Genetics
Nutrition
Environmental factors
2
Cellular and Molecular Biology
Prokaryotic and Eukaryotic Organisms
Eukaryotic cell organelles
The Cell Cycle
The Central Dogma of Cell Biology
Eukaryotic genes
Ribonucleic Acid
Messenger RNA and translation of protein
RNA polymerases
Regulation of gene transcription
Transcriptional control of specific genes
Transcription factors
Biotechnology and Genetic Engineering
Transgenic Animals and their Potential
Identifying, Isolating and Transferring Genes
Homologous recombination, gene targeting
and gene knockouts
3
The Endocrine System
Control of Pituitary Hormone Secretion
Hormone Receptors
Effector Molecules, Transducers and Second Messengers
1
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3
4
6
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9
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48
v
vi
Contents
4
Development of Muscle, Skeletal System
and Adipose Tissue
Embryonic Development and Cell Differentiation
Cell Proliferation and Differentiation
The Skeletal System and Bone Growth
Types of Bones and Gross Anatomy
Bone Cells and Bone Matrix Formation
Osteoclasts and bone resorption
Regulation of osteoclast function
The Epiphyseal Plate
Skeletal Muscle Development
Embryonic formation of skeletal muscle
Skeletal muscle satellite cells
Myofilaments, Myofibrils and Sarcomeres
Sarcomeric proteins: actin, myosin and titin
Myofibres and muscle contraction
Skeletal Muscle Fibre Types
Skeletal muscle protein turnover
The formation of multinucleated skeletal muscle cells
Adipose Tissue Growth and Development
Brown adipose tissue
White adipose tissue
Adipose tissue development
Adipose tissue growth: hyperplasia vs hypertrophy
Lipogenesis and Lipolysis
5
Growth Hormone and Insulin-like Growth Factors
The GH Molecule
Control of GH secretion
GH receptors
Metabolic actions of GH
Excess and inadequate GH: giants and dwarfs
IGFs or Somatomedins
Discovery of the IGFs
IGF genes and molecules
IGF receptors
IGF-binding proteins
Metabolic effects of the IGFs
Effects of the IGFs on animal growth
Modification of the somatomedin hypothesis
Effects of GH on Farm Animal Production
GH and the development of transgenic animals
6
Calcium Homeostasis
and Regulation of Bone Growth
Hormonal Regulation of Calcium Homeostasis
55
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124
Contents
7
Parathyroid hormone
1,25-Dihydroxy vitamin D3
Biological actions of 1,25-dihydroxy vitamin D3
Calcitonin
Regulation of Bone Growth and Development
The hedgehogs and early skeletal differentiation
Regulation of bone growth and differentiation
by hormones
Growth factors and bone
FGF
PDGF
Transforming growth factor-β
Bone morphogenetic proteins
124
126
127
129
131
131
Hormones, Growth Factors and Skeletal Muscle
146
146
147
148
150
151
153
153
155
156
156
157
157
158
Experimental Systems to Study Myogenesis
Myogenic cell systems
Muscle Regulatory Factors (MRFs)
Effects of the MRFs on muscle development
Regulation of MRF expression
Growth Factors Affecting Muscle Growth
IGFs
Myostatin
FGF
TGF-β
BMPs
Hepatocyte growth factor
Hormones regulating muscle growth
8
vii
Hormones, Growth Factors
and Adipose Tissue
Cell Systems Used to Study Adipogenesis
Transcription Factors and Adipogenesis
PPARs
C/EBPs
ADD1/SREBP1
Nuclear co-activators and co-repressors: fine-tuning the
adipogenic response
Hormones and Growth Factors Affecting Preadipocytes
and Adipocytes
Insulin
GH and the IGFs
Glucocorticoids
Prostaglandins and cAMP
Negative Regulators of Adipose Differentiation
Adipose tissue as an endocrine organ
132
135
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137
140
143
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163
166
167
169
170
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173
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176
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viii
Contents
9 Steroids and Animal Growth
Oestrogens and Ruminant Growth
Oestrogens and Non-ruminants
Androgens and Ruminant Growth
Mechanism of Action of Steroids in Ruminant Growth
Androgen mechanisms of action
10 Catecholamines, Beta-agonists and Nutrient
Repartitioning
The Autonomic Nervous System and the Adrenal Medulla
Adrenergic Receptors
β-Adrenergic agonists
Effects of β-adrenergic agonists on growth
and body composition
11 Leptin, Body Composition
and Appetite Control
Theories on the Short-term Regulation of Food Intake
The Long-term Regulation of Food Intake – the Lipostat Theory
The Discovery of Leptin
The leptin gene and molecule
Leptin receptors and binding proteins
Biological actions of leptin
Effects of leptin on reproduction
Direct peripheral effects of leptin
Regulation of leptin concentrations
Significance of leptin to animal production
Appetite-inducing (Orexigenic) Peptides of the Hypothalamus
Index
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1
Whole Animal Growth
The primary focus of this book is to examine the regulation of farm animal
growth by hormones and growth factors, primarily at the cell and molecular
level. As such, most of this book will be devoted to the study of developmental, cellular and molecular mechanisms that are involved with the regulation of cell and tissue growth. This chapter is designed to provide a cursory
outline of some of the most basic aspects of whole animal growth. I hope
that this chapter will inform readers of the processes involved with animal
growth and expose them to some of the factors that influence growth and its
measurement at the organismal level.
Measuring Animal Growth
The study of animal growth can be undertaken at several levels, ranging
from the cellular and molecular to whole animal studies. At the cellular
level, growth may be quantitated by measuring the increase in cell size and
cell number. This can be done directly by counting numbers of cells or by
measuring changes in their size using a microscope. We can also measure
cell number or mass indirectly, using chemical analysis. As the amount of
DNA in a cell is constant, quantitation of DNA concentration per unit mass
can be used as an accurate reflection of cell numbers. In addition, the use of
the other primary cell macromolecules, RNA and protein, can be measured
as a reflection of cell, tissue or organ mass. These are somewhat less accurate
than using DNA measurements, as the amount of protein and RNA may
change as a result of experimental treatment. At the organ level, changes in
organ mass or function can be quantitated during different developmental
stages or in response to experimental treatments. At the whole animal level,
growth parameters can be quantitated during different portions of the life
cycle. The study of growth can focus on changes that occur during embryonic and fetal growth, or during the postnatal period that occurs immediately after parturition. Some studies are focused on pre-pubertal growth, the
time between birth and puberty. Of interest in animal husbandry is the ‘fattening’ phase of adult animal growth, when farm animals have essentially
ended their linear growth but continue to accumulate fat, altering their body
©K.L. Hossner 2005. Hormonal Regulation of Farm Animal Growth (K.L. Hossner)
1
2
Chapter 1
composition and the characteristic flavour of the meat derived from these
animals.
At the cell level, growth can occur in two ways. Cells can replicate via
the process of mitosis to increase overall mass by increasing cell numbers.
This process of cell replication is called hyperplasia. The second way in
which cells can grow is to increase in size or volume. This process is
known as hypertrophy. During the formation of organs during embryogenesis and in cells grown in tissue culture, cells initially increase their numbers
by mitotic replication, cease replication and then grow by expanding in size.
The growth in size or volume of cells is accompanied by protein synthesis,
and in the case of adipocytes, by accumulation of large amounts of lipid.
Cellular hypertrophy is especially prominent during the development of the
large cells characteristic of mature adipose tissue and skeletal muscle. This
postmitotic alteration in cell size and shape is accompanied by qualitative
alterations in gene expression. As a result, cells change from relatively simple, proliferating cells into differentiated cells that assume specialized functions associated with the mature organ. For example, the development of
specialized intracellular structures such as contractile fibres in muscle or
intracellular accumulation of large amounts of lipid that serves as energy
storage depots in adipocytes.
Animal growth is a quantitative factor that can be measured objectively. In the living animal, as bones lengthen during linear growth,
changes in body weight, height or length accompany growth. In addition,
qualitative changes occur in body proportions and functions, which
accompany maturation and the increase in body size. The study of the
changes in body proportions during growth is called allometry. At the
whole animal level, growth is measured by an increase in length of bony
appendages (height or length), or the enhanced growth of muscles and
organs. Quantitative increase in muscle mass is important for animal
producers and ultimately, consumers. The quality of muscle growth is
also of importance for the production of meat for consumption and the
ratio of intramuscular fat to muscle is an important measure of meat
quality.
The simplest and most commonly used method to measure whole animal growth is to simply measure body weight. But when doing this, one
must be aware that simple body weights may reflect water accumulation,
gut fill, fat deposition, as well as wet hide weight and faecal contamination, not necessarily true growth. Thus, it is important to obtain body
weight of different animals under identical conditions of husbandry, so
that effects of feeding and watering, as well as transportation, are eliminated. Animals, which are transported over long distances, in some cases
for many days, arrive at their destinations dehydrated and underfed.
Rapid rehydration by excess water consumption leads to falsely high body
weights. Body weights for cattle obtained after 24 h without feed, but with
access to water, provide a ‘shrunk body weight’, which is highly correlated
with empty body weight, i.e. the weight of the body minus the gut
contents.
Whole Animal Growth
3
Embryonic and Fetal Growth
Growth is composed of two basic components that occur sequentially, but
overlap over time. As discussed earlier, true growth consists of an increase
in mass, length and height of an animal. At the same time there is a change
in body form or proportions. This is due to the differential growth of tissues
and is most evident in fetal development. As shown in Fig. 1.1, the changes
in body proportions that accompany growth from the human fetus to the
adult change dramatically. During development, the head and brain develop
first and in the early fetus, the head occupies one-half of the body length,
while in the adult, it accounts for about one-eighth of the body length. In
contrast, the hind limbs develop later, occupying only an one-eighth of the
body length in the fetus, but roughly one-half in the adult. Changes in body
proportions or form also play a role in the ultimate body shape of farm animals and form the criteria for judging animals in the show ring and determining fat content in the feedlot. In mature humans this is best seen in
sedentary individuals who, as a result of differential fat deposition, adopt an
apple or pear-shaped body form over time. Development of optimal proportions of the major tissues involved with animal production, muscle, bone
and fat, are essential to produce an animal which is economically efficient to
manage and produces a relatively large proportion of meat which is acceptable to the consumer. A high amount of muscle with a minimum amount of
bone and fat is an ideal condition for meat animals. The most efficient animals are those that rapidly gain weight and reach a mature body size with
less body fat. Cattle being fed during the feedlot phase of production have
essentially ceased linear growth and now accumulate subcutaneous fat and
demonstrate the altered proportions of a relatively sedentary animal.
Fig. 1.1. Changes in body proportions during development. Adapted from Stratz (1909).
4
Chapter 1
During embryonic growth, tissues grow sequentially (Fig. 1.2), proceeding from anterior to posterior during development. In early embryos the
components of the central nervous system (CNS), the brain and spinal cord,
develop first. Bone formation follows shortly thereafter and overlaps CNS
development. The bones form the scaffolding around which muscle cells
form and attach during fetal development. Adipose tissue is the last tissue to
develop. Adipose tissue is formed primarily during postnatal development,
although some adipose tissue primordia are formed during embryogenesis
in many species. Adipose tissue is primarily a tissue of maturity, which is
used to store excess energy in the form of fat, or triglycerides. This occurs in
the postnatal period when nutritional energy is abundant and is in excess of
the body’s requirements for energy utilization.
Growth Curves
Whole animal growth can be described in several ways using different types
of growth curves (Fig. 1.3). Probably the most common way of describing
growth is to simply plot height or weight of the animal vs time (Fig. 1.3A).
This results in a sigmoidal curve that is characteristic of the growth of many
living organisms, including animals and their organs and plants and their
components. Bacterial colonies and animal cells grown in vitro also follow
sigmoidal growth curves. The sigmoidal growth curve is characterized by an
Fig. 1.2. Growth rates of body regions and tissues during development. Adapted from
Hammond (1955).
Whole Animal Growth
5
Fig. 1.3. Types of growth curves.
initial exponential growth phase, when growth is very rapid and the slope
of the curve is maximal. The exponential phase is followed by an inflection
point when the shape of the curve changes from the very rapid growth of the
young organism to a more moderate, slower growth rate. Finally, the growth
rate reaches the growth plateau, when growth is very slow and essentially
ceases.
6
Chapter 1
Another type of growth curve is derived by plotting the average daily
weight gain against the age of animal (Fig. 1.3B). This curve emphasizes the
rapid growth rates seen during the postnatal period, culminating in the high
growth rate during the time of maturation, followed by a slow decline in
growth rate until mature size is reached. Fig. 1.3C shows the per cent increment growth curve. In this curve, the percentage increase in body weight is
compared to the previous weight. This curve emphasizes the extremely
rapid growth of the early fetus – over a 1000% rate – which rapidly declines
in the postnatal animal.
Genetics, Nutrition and Environmental Effects
on Whole Animal Growth
The most important of the factors that control whole animal growth are the
animal’s genetic background, its nutritional intake and the environment that
the animal is exposed to. These factors have provided the fundamental basis
for the traditional control and manipulation of animal growth throughout
the history of animal agriculture. They are the most obvious and visible variables that affect the growth of all organisms, including humans, which can
be controlled to optimize the production of farm animals. Optimal growth,
production and health of animals depend upon an animal with good genetic
qualities that are reflected with the phenotype of the animal. Likewise, animals must be provided with adequate nutritional resources in order to survive and produce a product of quality. Animals maintained in a sheltered
environment that is protected from extreme temperature and climatic fluctuations will perform better than those that are not. As a major proportion of
the animal sciences curriculum focuses on these factors, these topics will be
discussed only briefly. One should keep in mind that hormones of the
endocrine system are influenced by all of these factors: genetics, nutrition
and environmental stresses. As a rule, these parameters are under rigid
experimental control when endocrine studies are carried out.
Genetics
The genetic background of an animal provides the fundamental capacity for
rapid growth, body composition and adult size. The genetic background of
an animal encompasses all heritable traits. These include such things as coat
colour and density, bone structure, growth rates, muscling and fat deposition. While some of these characteristics are simple, single gene-mediated,
dominant or recessive traits, most inherited traits of interest to animal producers are the result of the effects of multiple genes working cooperatively.
These traits, called quantitative traits, are the cumulative result of tens or
hundreds of genes working together. The genetic background of an animal
provides the basic potential for ultimate growth rate and mature size (height
and weight).
Whole Animal Growth
7
Characteristics of animal growth are heritable and contribute to the large
variation seen in animal body sizes and growth rates. One only has to look
at the wide variation in the shape and size of dogs to see the effects of genetics and genetic selection, on morphology and body size. Dog breeds range in
size from a few ounces in Mexican Chihuahuas to a few hundred pounds in
Saint Bernards. The long-legged, lean body form of racing breeds such as the
greyhounds and whippets is in stark contrast to the short, pug-faced
English bulldogs or the small, short-legged Dachshunds. The heritability of
growth characteristics accounts for 25% to 30% of the variation in growth.
Some of the heritable traits can be attributed to major gene effects and single
genes have been implicated in several instances in which growth or body
composition are altered. The double-muscling phenotype of Belgian blue
and Piedmontese cattle that actually results from muscle hypertrophy is due
to a mutation of the myostatin gene. The naturally occurring myostatin gene
acts to limit muscle growth and cattle inheriting the mutant inactive form
undergo significant muscle hypertrophy associated with the doublemuscling phenotype. Callipyge sheep are also characterized by muscle
hypertrophy, but in this case, hypertrophy is localized to the hind limbs and
sheep with this phenotype have large hindquarters (callipyge means ‘beautiful buttocks’ in Greek). This distinct alteration of morphology is due to a
single non-recessive gene inherited from the sire.
In contrast, most complex phenotypic traits, such as the graded, continuous variation seen in animal body size, growth rates and body composition
result from the cooperative effects of many genes, each with a variable contribution to a specific complex phenotypic trait. These multigene effects are
called quantitative traits and are the primary focus of breeding and selection
in animal agriculture. In sheep and cattle, selection has centred on body
weight at a specific age, either weaning weight in sheep or weaning, yearling or 18 months weights in cattle. Rate of growth, measured as average
daily gain (ADG), is often used as a selection criterion in farm animals. In
contrast to cattle and sheep, pigs have a relatively short generation time and
produce multiple offspring, making them more amenable to selection studies. Selection for higher growth rates in swine over ten generations leads to
rapid improvements in production characteristics. Growth rates, measured
as ADG, can be increased by almost 50%, while feed intake is increased by
about 35% and efficiency of feed utilization increased by about 13%.
The advent of molecular biological methods has allowed the use of
molecular genetic markers as selection criteria in farm animals. Two general
types of genetic markers are used for improvement of production characteristics, single locus markers and multiple locus markers. Single locus markers
represent a single gene of particular significance, one that is associated with
major production traits, such as the genes for the hormones leptin, growth
hormone (GH) or insulin-like growth hormone-I (IGF-I). These hormones
are associated with body composition, growth rates and birth and weaning
weights. As such, they exert effects on multiple organ systems including
bone, muscle and fat as well as the overall energy metabolism and nutrient
partitioning in the body. At the molecular level, genes for single hormones
8
Chapter 1
exist as molecular alleles that have variable overall lengths, as measured in
the number of bases of DNA that comprise a specific gene. These molecular
alleles may confer tissue specificity of gene expression or may result in a
gene product that has enhanced biological activity. In addition, alterations in
the sizes or numbers of gene regulatory elements associated with specific
alleles may increase or decrease the response of a gene to factors that regulate the activity of a certain gene. These single gene alleles may be studied at
the molecular level using restriction fragment length polymorphism
(RFLP). These fragments of DNA from specific genes are generated using
restriction endonucleases, which cut the gene at specific sites, or by PCR
amplification of specific gene sites. This generates a set of two or three RFLP
fragments of the gene, which represent the molecular alleles. The RFLPs can
then be used as selection markers. The use of RFLPs as genetic markers provides limited information from only a single gene and the use of multiple
single gene markers in a single individual or breed is more likely to provide
useful selection information. To date, the use of single gene markers to
improve production characteristics has not proven to be very useful for
selection studies.
As hormones are present in the circulation, another approach has been
to use genetic selection based on systemic hormone levels, with the
assumption that gene activity is reflected by concentrations of hormones in
the bloodstream and that a single hormone can effectively alter growth,
growth rates and metabolism. The utilization of hormones as genetic markers must be done with the knowledge that hormone concentrations are
influenced by many variables other than genetics, including developmental stage, breed, nutrition, time of day or year and so on. The earliest hormones used for this approach included insulin and thyroid hormones,
which are related to growth and differentiation. Due to the unwanted metabolic side effects of these hormones (e.g. abnormal lipid, carbohydrate and
protein metabolism), their use as selection tools has not been fruitful.
Another hormone used for this type of analysis was GH, which is associated with growth rate and lean tissue growth. This use of GH in selection
studies (and all studies) is complicated by the fluctuations in serum GH
concentrations seen on a minute-to-minute basis. To accurately measure
GH, blood samples must be drawn at 10 to 15 min intervals for a 6 to 8 h
period. As GH concentrations are also affected by stress and nutrient
intake, these variables must also be carefully controlled. Results of selection
based on GH concentrations have been inconsistent and animals selected
for increased levels result in either fat deposition or leaner phenotypes.
IGF-I is a hormone that mediates the effects of GH. Unlike GH, IGF-I serum
concentrations are constant and single blood samples are adequate to establish circulating concentrations. Concentrations of IGF-I are correlated with
body weight and growth rate in sheep, cattle, pigs and chickens. Selection
based on IGF-I concentrations in Angus beef cattle over a 10-year period
has shown that elevated serum IGF-I concentrations are only weakly associated with backfat thickness or muscling in postweaning bulls and
heifers.
Whole Animal Growth
9
Multiple locus markers are genetic markers that occur in multiple sites
throughout the genome. An ideal multiple locus marker is one that is spread
throughout the genome, occurring at regular, equal distances on all chromosomes. The multiple locus markers used for molecular markers are highly
polymorphic, existing as several different sizes of repetitive DNA fragments,
for example, [-GT-]n, [-GACA-]n or [-CTCTGGGTGTGGTGC-]n. The presence of these variable tandem repeats is revealed by treatment of isolated
DNA with restriction endonucleases or by amplification of specific
sequences using PCR. In contrast to single locus markers, multiple locus
markers do not code for proteins. These repetitive sequences are found in
regions of DNA called satellite DNA, either as mini-satellite DNA, consisting of repeats of two to ten bases, or micro-satellite DNA, which consists of
16 to 33 base repeats sequences. The multiple locus markers are used in linkage analyses to examine the inheritance of quantitative trait loci (QTL). QTL
are composed of many genes that work in concert to regulate complex production traits such as growth rate, milk and wool production or body
composition. Association of QTL with specific multiple locus markers provides a method to indirectly examine the heritability of these complex multigene characteristics. Multilocus marker patterns are specific to individuals
and are used as DNA fingerprints to identify individuals, pedigrees, paternity and genetic diseases.
Nutrition
A great deal of time, effort and money has been spent studying the nutritional requirements of animals and the effects of nutrients on animal growth,
composition and production. Animal nutrition is a major focus of the animal
science curriculum and only cursory comments are given here. Providing
the optimal amounts of nutrient to fulfil an animal’s requirements results in
a healthy animal with good production characteristics. In the context of a
specific genetic background, adequate nutrition is required to obtain optimal
growth and reach the animal’s genetic potential. Adequate amounts of protein, carbohydrate, fat, vitamins and minerals are needed to attain the full
genetic potential of an animal. In addition, specific growth promoters such
as iodine, calcium, zinc, cobalt and vitamins, as well as the essential amino
acids lysine and tryptophan and the essential fatty acids linoleic, linolenic
and arachidonic acid are required for optimal growth of animals. The
amount and quality of feed is the most important factor in regulating growth
rate. Growth is halted or retarded by limiting feed intake. This is, in general,
a reversible process and growth will resume if adequate food is returned to
the animal. However, if severe nutritional inadequacies are present during
early development, permanent stunting of animal size will result. Total
available energy intake is the most important dietary component in growth
variation. Energy intake can be altered by poor animal health, environmental factors and micronutrient availability. Specific nutrient deficiencies, for
example zinc or sodium deficiency, will reduce the feed intake of animals.
10
Chapter 1
Environmental factors
The primary environmental factors that affect animal growth and development are temperature extremes, either heat or cold, and photoperiod effects.
While other factors such as arid or humid climates and high altitudes also
affect animal health and production, they will not be considered in this section. Photoperiod can be defined as the biphasic change in light intensity or
the hours of light vs dark to which an animal is exposed on a daily basis.
Photoperiod varies with latitude and with yearly seasons and is considered
the most reliable and effective indicator of seasonal changes. In wild animals, the photoperiod serves to synchronize nutrient availability with foraging activity and nutrient use. In addition, seasonal changes in day length
induce seasonal breeding to ensure that parturition occurs at an appropriate
time of year, usually in the springtime. Most photoperiod effects on breeding and growth have been minimized in domestic animals such as cattle and
pigs owing to their intensive breeding and selection for optimal growth and,
in the case of swine, the use of artificial housing and lighting. Small ruminants, such as sheep and goats, are still strongly seasonal animals whose
growth and reproduction are regulated by photoperiod.
In general, increasing the length of time an animal is exposed to light, up
to a point, enhances feed intake and growth rates. Increasing light exposure
from the normal 8 to 10 h/day to 14 to 16 h increases growth rates by up to
10% in cattle, sheep and poultry when they are maintained under controlled
environmental conditions. Continuous exposure to light (23–24 h) has detrimental effects on growth and production. Longer day lengths have no effects
on feed consumption or growth rates in pigs. In sheep and cattle, the primary benefit of longer periods of light exposure is to reduce carcass fat while
increasing carcass protein.
The differences in an animal’s thermal environment have significant
effects on growth and production. The wide disparities in climatic environments between the temperate zones and those of the desert, the northern latitudes and equatorial regions provide a wide variety of climates. Many of
the problems encountered with farm animals involve the introduction of
European breeds of dairy and beef cattle into extremely hot or cold environments. When domestic animals are exposed to extremes of heat or cold, they
must expend a greater amount of energy to maintain their body temperatures. This diverts energy away from growth and from the production of
meat, milk and progeny. Animals perform best when maintained within a
thermoneutral temperature range. This is the temperature at which there is
only a minimal amount of body heat loss or gain. While the thermoneutral
range varies by species a general comfort zone ranges from 20°C to 25°C.
When temperatures are below this range, animals must conserve and generate heat, while at temperatures above the thermoneutral range, animals
must cool themselves by physiological and behavioural adaptations.
The effects of cold temperatures, or cold stress, are especially pronounced in neonatal animals. Exposure of the newborn to low temperatures
during the first 72 h of life greatly increases the morbidity and mortality of
Whole Animal Growth
11
these animals. Cold stress and the accompanying reduced nutrient intake
can increase neonatal mortality rates by 25% to 50% and is obviously an
important factor in reproductive success. Animals that are smaller at birth
are more susceptible to cold stress due to their reduced capacity for thermogenesis (heat production). Smaller animals also have a relatively larger surface area in relation to their body weight, providing a greater exposed area
for heat loss. Thus, twin and triplet lambkins and runt piglets are especially
susceptible to cold stress.
The effects of cold stress on growth and production characteristics are
especially prominent in swine. Perhaps the most obvious behavioural
response to cold stress is an increase in food consumption. This provides the
energy for metabolism and thermogenesis so that an animal can maintain a
constant body temperature in a cold environment that fosters heat loss. A
reduction of external temperature of only 1°C is associated with an increase
of 1% to 1.5% of feed intake in pigs in the temperature range of 10°C to 25°C.
Although the growth rates of domestic pigs are unaffected by temperatures
between 8°C and 20°C, increased feed intake reduces the efficiency of feed
utilization. While cold stress is, perhaps, less evident in large ruminants, it
has a significant effect on feed consumption. For example, feedlot cattle consume 20–30% more feed during winter months in northern latitudes than in
summer months. This is accompanied by similar reductions in feed efficiencies (−27% to 37%) and growth rates (−22% to 33%).
Cold stress induces a number of behavioural and physiological changes
that result in body heat conservation and heat production. In addition to
increased food consumption, peripheral vasoconstriction redirects blood
from the periphery to the body core. This retains heat and reduces heat loss
due to passive evaporation and heat exchange with the environment.
Animals exposed to cold temperatures huddle together to share body heat
and reduce exposed surface areas. Hair coat thickens and hairs stand erect,
while birds fluff their feathers as additional barriers to heat loss. Shivering
generates additional heat. Long-term exposure to cold results in anatomical
changes. Pigs reared at 12°C develop a more squat, rounded morphology
with shorter bodies, limbs, snouts, tails and ears. Fat depots undergo redistribution as the percentage of backfat is increased and internal (leaf) fat is
reduced. These morphological and anatomical alterations reduce heat loss
by providing insulation and by reducing surface area. The generation of heat
production in cold-stressed animals is enhanced by mobilization and metabolism of energy substrates. In addition to enhanced food intake, endogenous
energy substrates, in the form of glycogen and fat, are utilized to provide
energy for metabolism and heat production.
Elevated environmental temperatures present problems to the animal that
mirror those of cold stress. Heat stress problems are especially prominent in
European breeds of cattle that have been imported to the hot climates of the
tropical, subtropical or equatorial regions of the world. In contrast to coldstressed animals, in which food consumption is increased, the most important
response to heat stress is a reduction in food consumption. This reduction in
energy substrate availability results in a reduction of metabolism and
12
Chapter 1
utilization of dietary protein. This leads to a protein catabolic rate that exceeds
protein synthesis and results in a negative nitrogen balance in the body.
Heat stress generally impairs animal growth characteristics. Growth
rates, live weights and dry body weights are all reduced in response to heat
stress, while body water content is elevated. As a result of reduced food
intake, there is a reduction in anabolic metabolism and an elevated catabolism of fat depots and/or lean body mass. The upper critical temperatures
for European cattle are 24–30°C for growth and 21–27°C for milk production.
At 30°C, milk production is reduced by about 15%, while at 38°C it is
reduced by 30%.
Physiological responses to heat stress are characterized by increased
heart rates and peripheral vasodilatation that shunts blood from internal
organs to peripheral surfaces where heat is dissipated. This is accompanied
by hair and feather flattening for more efficient heat exchange. Evaporative
processes that enhance cooling, such as increased shallow respiration rates
(panting) to enhance evaporation of water from the lungs, passive evaporation from body surfaces and active water loss by perspiration, also play
important roles in dissipating heat. Behavioural alterations include a reduction in physical activity, enhanced water consumption and seeking coolness
in shade, water or mud.
References and Further Reading
Batt, R.A.L. (1980) Influences on Animal
Growth and Development. University Park
Press, Baltimore, Maryland, 60 pp.
Berg, R.T. and Butterfield, R.M. (1976) New
Concepts of Cattle Growth. Halsted Press,
New York, 238 pp.
Buttery, P.J., Haynes, N.B. and Lindsay,
D.B. (1986) Control and Manipulation of
Animal Growth. Butterworths, London,
347 pp.
Clutter, A.C., Jiang, R., McCann, J.P. and
Buchanan, D.S. (1998) Plasma cholecystokinin-8 in pigs with divergent
genetic potential for feed intake and
growth. Domestic Animal Endocrinology
15, 9–21.
Davis, G.P. and Denise, S.K. (1998) The
impact of genetic markers on selection.
Journal of Animal Science 76, 2331–2339.
Hammond, J. (1955) Progress in the Physiology
of Farm Animals, Vol. 2. Butterworths,
London, 740 pp.
Jefferies, A.J., Wilson, V. and Thein, S.L. (1985)
Hypervariable minisatellite regions in
human DNA. Nature 314, 67–73.
Lawrence, T.L.J. and Fowler, V. (2002) Growth
of Farm Animals, 2nd edn. CAB
International, Wallingford, UK, 368 pp.
Phillips, C. and Piggins, D. (1992) Farm
Animals and the Environment. CAB
International, Wallingford, UK, 430 pp.
Scanes, C.G. (2003) Biology of Growth of
Domestic Animals. Iowa State Press,
Ames, Iowa, 408 pp.
Stratz, C.H. (1909) Wachstrum und proportionen des menschen vor und nach der
geburt. Archives in Anthropology 8,
287–297.
2
Cellular and Molecular Biology
Current biology, whether it is the study of fruit fly genetics or animal science,
is often focused at the cellular and molecular levels. It is at this level that further advances in the improvement in production of animals are likely to
occur. Thus, a strong background in these concepts is essential to foster the
discovery of methods and processes that will alter animal growth, metabolism and production. This chapter presents an outline of the important concepts in molecular and cell biology that are required for an understanding of
the processes of animal growth control at the cellular level. While this information may be redundant for some students, it is my experience that many
students will benefit from this review. This chapter provides rudimentary
information about molecular and cellular mechanisms, with an emphasis on
eukaryotic cells.
Prokaryotic and Eukaryotic Organisms
Two broad categories of organisms encompass the range of life. Prokaryotic
organisms, the bacteria and archaeobacteria, are comparatively simple,
single-celled organisms. They differ fundamentally from the eukaryotic
organisms of fungi, plants and animals. Most eukaryotes are multicellular
organisms, although a few are single-celled. Multicellular eukaryotes are
composed of many different cell types which are specialized for different
functions and the cells of eukaryotic organisms are much larger than those
of prokaryotes. The structure and functions of eukaryotic cells are much
more complex than those of prokaryotic cells. The most distinguishing characteristic of eukaryotic cells is the presence of a nucleus (Greek: karyon
means kernel or nucleus) and other membranous organelles. Prokaryotic
cells have no distinct nucleus, while eukaryotic cells contain a nucleus that
is separated from the cytoplasm of the cell by a nuclear membrane. In addition, eukaryotic cells have distinct chromosomes composed of DNA that
forms a complex with proteins called histones. This combination of DNA
and protein is called chromatin. Prokaryotes, on the other hand, have a single, naked strand of circular DNA without the associated protein coating.
The DNA in the nucleus of eukaryotes consists of 107 to over 1010 base pairs
©K.L. Hossner 2005. Hormonal Regulation of Farm Animal Growth (K.L. Hossner)
13
14
Chapter 2
(bp), arranged in a linear fashion, interrupted by non-coding regions called
introns. Prokaryotic DNA is much smaller, containing less than 5 × 106 bp,
has no introns and is found in the cytoplasm. Finally, eukaryotic cells have a
cytoskeleton, composed of polymers of actin that form filaments and microtubules. These provide mechanical support for the cell and allow intracellular movement of organelles. Prokaryotes depend on a rigid cell wall, similar
to that of the plants, to support the cell and maintain its shape.
Eukaryotic cell organelles
In the eukaryotic cell, multiple intracellular structures made of membranes
are organized to form organelles that are absent in prokaryotic cells. The
presence of intracellular organelles in eukaryotes results in a compartmentalization of the cell and a consequent physical isolation of various cell
processes (Fig. 2.1). The plasma membrane surrounding the eukaryotic cell
consists of a hydrophobic lipid bilayer that forms a permeability barrier
around the cell. This prevents the free flow of ions and water-soluble molecules between the cell and its external environment. Consequently, molecules entering and leaving the cell do so under regulated processes. The
uptake and release of ions such as sodium and potassium are regulated by
energy-dependent transport processes, using ATP as an energy source.
These processes maintain cellular ionic balance and the electrical potential
across the cell membrane of the cell. Likewise, glucose enters the cell by a
transmembrane transport molecule. Of importance for hormones, the
plasma membrane also contains cell-specific transmembrane hormone
Plasma membrane
Free ribosomes
Rough endoplasmic reticulum
Nuclear
membrane
Nucleus
Nuclear pore
Smooth endoplasmic reticulum
Golgi complex
Secretory
vesicles
Peroxisome
Cytoplasm
Fig. 2.1. Eukaryotic cell organelles.
Mitochondrion
Lysosome
Cellular and Molecular Biology
15
receptors that act to bind circulating hormones, transduce their presence
through the cell membrane and activate cytoplasmic second messengers.
These messengers act as couriers of the hormone signal from the extracellular environment to the nucleus of the cell, resulting in an alteration of gene
expression and cellular metabolism.
Eukaryotic organelles are intracellular membranous structures with distinct functions that exist in multiple forms. The nucleus is segregated from
the cytoplasm by the nuclear membrane, which surrounds and separates the
genetic material (genes and chromosomes) from the cytoplasm. It consists
of a double membrane interspersed with nuclear pores that allow diffusion
of chemicals and communication between the nucleus and the cytoplasm. In
the cytoplasm, the endoplasmic reticulum is a closed system of sac-like cavities
and tubes made of a surrounding membrane. Rough endoplasmic reticulum
(RER) has many ribosomes attached to it, giving it a roughened appearance in
cells. This is where proteins are synthesized for insertion into membranes,
lysosomes or export from the cell. Smooth endoplasmic reticulum (SER) has no
associated ribosomes and synthesizes proteins for use within cells, either as
soluble nuclear or cytoplasmic proteins or membrane-associated proteins.
The SER is also the site of drug metabolism in liver cells and steroid synthesis in endocrine cells. The presence of a signal peptide determines whether a
protein is synthesized in the cytoplasm by free ribosomes or in the RER. The
signal peptide is an amino-terminal 16 to 60 amino acid extension of a protein.
This is the first part of the protein that is synthesized. All protein synthesis
is initiated in the cytoplasm on free ribosomes. If the signal peptide is present in the newly formed ribosomal-bound peptide, the peptide binds to a
signal recognition particle. The particle, the ribosome and its attached immature protein are translocated to the RER. The ribosome attaches to specific
receptors on the RER and the signal peptide passes through the membrane
of the RER, and leads the growing polypeptide into the lumen of the RER.
The signal peptide is cleaved within the RER and the protein enters lysosomal or secretory vesicles.
Like the endoplasmic reticulum, the Golgi apparatus is a network of
membrane-bound cavities and spaces, arranged in stacks within the cytoplasm. This is the site of protein maturation and protein sorting. The Golgi
apparatus receives newly synthesized proteins from the RER and, in a process
called post-translational modification, adds sugars to the protein chain to
form glycoproteins. The modified proteins are then transported in vesicles to
lysosomes, the plasma membrane or to secretory vesicles for release from the
cell. Lysosomes are vesicles that contain catabolic enzymes for hydrolysis of
macromolecules. Peroxisomes are membrane-bound organelles that contain
enzymes, which use oxygen as an oxidizing agent and produce hydrogen
peroxide as a by-product. They are responsible for detoxification of chemicals such as ethanol, phenols and formaldehyde. Peroxisomes are also
important in the degradation of long-chain fatty acids via the β-oxidation
pathway. Like mitochondria, peroxisomes reproduce by division.
Mitochondria are small, double-membraned organelles found in almost all
eukaryotic cells in large numbers. Mitochondria contain their own DNA,
16
Chapter 2
which is circular and not associated with histones. This DNA is used to synthesize a small proportion of mitochondrial proteins, although most mitochondrial proteins are derived from nuclear genes. Mitochondria provide
the metabolic energy, in the form of ATP, to cells and are most abundant in
cells with extensive oxidative metabolism. Mitochondria are the site of the
oxidative degradation of food within the cell. Oxidative phosphorylation,
the conversion of pyruvate to acetyl-coenzyme A (acetyl-CoA), the citric acid
cycle and the respiratory chain that produces ATP, all occur in the mitochondria. Fatty acid degradation, via the process of β-oxidation, occurs in
the mitochondria. Mitochondria are capable of self-replication and are inherited by offspring only from the maternal parent.
The Cell Cycle
As a eukaryotic cell undergoes mitosis, it must accomplish two things. It
must first double the amount of DNA in its nucleus and then it must halve
this amount of DNA. These processes occur as the cell passes through
defined functional states, called the cell cycle (Fig. 2.2). In all, cell division
in mammals takes anywhere from 10 to 24 h depending on the cell. A 24-h
cell cycle will be used in the example given here. The two basic portions of
a cell’s life are mitosis (M) and interphase. Mitosis entails the formation of
microscopically visible chromosomes and their separation into daughter
cells. Due to the visible nature of the chromosomes, mitosis was once the
focus of many cell studies and has been well documented. While important,
mitotic events are only a small part of the cell’s life cycle. What was once
called the ‘resting’ phase, or interphase, when the chromosomes are not visible, is now known to be the time at which cell metabolism and specialized
functions occur. The events that occur during interphase are now called the
cell cycle. The cell cycle consists of distinct portions in which DNA synthe-
Fig. 2.2. The cell cycle.
Cellular and Molecular Biology
17
sis and the doubling of DNA content occurs, organelles are replicated and
attainment and expression of the differentiated state of the cell are apparent.
M: In this phase the formation and separation of visible chromosomes and
production of daughter cells occurs. Mitosis is divided into the sequential
phases, prophase, metaphase, anaphase and telophase, in which the chromosomes condense, pair and separate. This is followed by cytokinesis, the
separation of the cytoplasm and the formation of two daughter cells. In cells
grown in vitro, mitosis lasts about an hour.
G1: Originally called a ‘gap’ phase between mitosis and the S phase, this is
the first cell growth phase after mitosis. In this portion of the cell cycle, the
cell grows and carries out normal metabolism. This is usually the longest
and the most variable part of the cell cycle. It is during this phase that
organelles are duplicated. If cells are deficient in nutrients or mitotic signals
are absent, this portion of the cell cycle will be prolonged or the cell may
enter the G0 phase and undergo differentiation. In growing cultured cells,
this phase lasts 10 h.
G0: This is the phase in which the cell is ‘withdrawn’ from the cycle, characterized by active protein synthesis and, in some, motility. This is the phase in
which differentiated cells exist. It may last indefinitely or, under appropriate
conditions, such as stimulation by mitotic growth factors, the cells may
re-enter the cell cycle in the G1 phase to undergo mitosis.
S: The synthetic phase, when DNA synthesis and chromosome duplication
occurs. This phase lasts 8 h.
G2: This is the second gap, or growth, phase in which the cell grows and
prepares for mitosis. Newly replicated DNA is checked for damage and
errors in duplication and repaired if necessary. This phase lasts 12 h.
The cell cycle is under the control of regulatory proteins that are activated
and inactivated at specific times during the cycle. These regulatory proteins
are enzymes called kinases, which phosphorylate specific proteins, and
phosphatases, which remove phosphate groups from proteins. The kinases
are combinations of two different subunits that are activated by associating
with one another to form heterodimers. The regulatory subunit of this heterodimer is called cyclin and the catalytic subunit is called a cyclin-dependent
kinase, or Cdk. Concentrations of Cdks are relatively constant throughout
the cycle, but cyclin concentrations fluctuate with the phase of the cycle.
Thus, the abundance of specific regulatory subunits of the kinases determines the activity of these enzymes. After a specific phase of the cell cycle is
completed, the cyclins are dephosphorylated and degraded by the ubiquitin
proteolytic system of the cell. There are specific cyclins and Cdks for each
stage of the cell cycle. Cyclin D and Cdk4 are present in the G1 phase and initiate the preparation of chromosomes for replication. Cyclin A and Cdk2
prepare the cell for DNA synthesis of the S phase. In the G2 phase, the mitotic
cyclins, cyclins A and B, associate with Cdk1 to initiate events associated
with the beginning of mitosis, such as chromosome condensation and
nuclear membrane breakdown.
18
Chapter 2
The Central Dogma of Cell Biology
The central dogma of cell biology is the fundamental basis of cell and molecular biology. This can be summarized by three tenets: (i) DNA can self-replicate (semiconservative replication); (ii) DNA acts as the template for RNA
formation (a process called RNA transcription); and (iii) mRNA acts as the
template for the synthesis of protein (a process called protein translation).
These processes are outlined in Fig. 2.3.
The central dogma of cell biology forms the basis of all processes that
occur within the cell, and hence, within the living organism. It includes the
replication of DNA so that new cells can be formed, the synthesis of mRNA
from specific genes and the translation of the genetic message in RNA into
proteins that serve as functional macromolecules within the cell and as messengers between cells. In addition, factors external to the cell, such as hormones and nutrition, as well as innate, endogenous genetic messages, can
modulate any of these processes. Regulation of these fundamental processes
stimulates mitosis, cell enlargement and accumulation of protein. Altering
gene expression of mRNA and subsequently, protein, results in changes in
the amount of enzymes and other active proteins present in cells. It is also
the basis for the synthesis of proteins for export from the cells, such as
secreted factors that regulate the function of other cells, hormones and
growth factors. Accumulated proteins and secreted proteins alter the metabolism and thus, function of cells at a particular instant in time or over long
periods of time.
Fig. 2.3. The central dogma of cell biology.
Cellular and Molecular Biology
19
Eukaryotic genes
The function of a structural gene encoding for a specific mRNA is simply
to produce a protein. Proteins are diverse molecules, which play many
important roles in the cell. As enzymes, proteins catalyse all chemical reactions in the organism. Proteins may act as receptors that mediate hormone
actions. Proteins can also be hormones, which modulate the body’s response
to nutritional, environmental and genetic factors. Structural proteins such as
collagen, actin and myosin provide the shape and locomotion for cells and
organisms. Proteins are thus essential for the maintenance and regulation of
functions in the living organism. The unique sequence of amino acids that
defines a protein is coded by the heritable genes in chromosomes of the
nucleus. In turn, the overall three-dimensional structure of proteins, which
determines their biological role in the animal, results from the unique
amino acid sequence of the protein chain. Thus, the properties of these
important regulatory and functional molecules are embedded in the genes
of an organism.
The genes of eukaryotes are contained within the chromosomes of these
organisms. The chromosomes are densely packed structures consisting of
DNA and associated proteins, including histones and other proteins. These
proteins provide regulatory functions and serve to physically condense the
genetic information into a compact form that can be contained within the
nucleus. There are about 3 billion pairs of nucleotides (bp) in the human
genome, arranged into 46 chromosomes. Each chromosome contains a single
molecule of DNA that stretches from one end of the chromosome to the
other. Only 5% of this DNA codes for the protein products of expressed
genes. This is in contrast to prokaryotes, in which 90% of the DNA codes for
proteins. Much of the non-coding DNA in eukaryotes consists of repeated
sequences of nucleotides, called interspersed DNA and satellite DNA. The
function of non-coding DNA is largely unknown. It may do nothing specifically, simply representing the accumulation of innocuous nonsense DNA
over millions of years that is tolerated as long as it does not interrupt the
function of essential protein-coding genes. Some non-coding repetitive DNA
may play an as yet, unknown regulatory function, altering the expression or
silencing of protein-coding genes. Table 2.1 shows the sizes of the genome in
various species. The size of an organism’s genome varies widely and has no
relationship to the perceived complexity of the organism. The mammalian
cell contains 5 to 10 pg of DNA, consisting of 3 to 5 billion bp of nucleotides
and roughly 30,000 to 50,000 genes.
Genes are transcribed (rewritten) into RNA by enzymes called RNA
polymerases, which move from the 5′ to the 3′ end of the DNA. While a
prokaryotic gene that codes for a specific mRNA consists of continuous coding sequence of DNA, eukaryotic genes coding for mRNA are discontinuous, with large sections of interspersed non-coding DNA (Fig. 2.4).
Eukaryotic genes are composed of stretches of DNA called exons (expressed
regions) that encode for mRNA. Non-coding sections of DNA, called introns
(intervening regions), separate exons from one another. Introns can range in
20
Chapter 2
Table 2.1. Sizes of the genome in base pairs (bp) and number of chromosomes of various
species.
Species
bp
Human
3.5 × 109
23
Bovine
3 × 109
60
Mouse
3 × 109
40
Chicken
2.1 × 109
78
Frog
4.5 × 1010
26
5 × 1010
10
Maize
Chromosomes
Drosophila
1.6 × 108
8
Saccharomyces
cerevisiae (yeast)
1.4 × 107
34
4 × 106
1
Escherichia coli
size from 75 to 10,000 nucleotides. The average mammalian gene contains
eight introns, but can contain more than 50 of these non-coding regions
within a gene. Some of the non-coding intron regions play a part in the regulation of gene expression. A promoter region, located upstream (5′) to the
gene to be transcribed, consists of specific sequences of DNA to which RNA
polymerase II and other protein factors needed for gene transcription bind.
This is the area where gene transcription is initiated. The 5′ and 3′ flanking
regions of the gene contain distant regulatory elements that bind protein
transcription factors that enhance or suppress gene expression.
When genes are transcribed into mRNA, both introns and exons are
copied. The introns are then removed with specific enzymes by a process
called pre-mRNA splicing. This results in a mature mRNA that contains only
exons that is translated into protein (see later). The interrupted genes of
eukaryotes provide a way in which multiple mRNA and protein products
can be produced from a single gene, increasing the number and diversity of
Fig. 2.4. Eukaryotic gene structure. The 5′ and 3′ flanking regions and the 3′ untranslated
region contain enhancer regions and response elements that regulate gene activity.
Cellular and Molecular Biology
21
proteins produced by a gene. Many genes contain more than one promoter
site where gene transcription can begin. This results in some protein products that may be slightly larger (or smaller) than others. In addition, after
transcription, different combinations of exons may result due to alternate
splicing of the mRNA. That is, differential splicing of exons to one another
can result in the formation of multiple mRNAs from a single gene. Thus, a
gene may produce an mRNA that consists of, for example, exons 1, 2 and 3
or exons 1, 2 and 4. The resulting proteins are closely related isoforms of one
another, but one form may have enhanced biological activity or may appear
only in a specific tissue.
Ribonucleic Acid
RNA has many functions in the cell. Messenger RNA contains the code for
the amino acid sequence of the mature protein, transfer RNA carries specific
amino acids that are used in protein synthesis and ribosomal RNA provides
the catalytic centre of protein synthesis on ribosomes. The sizes of the RNAs
are often given as sedimentation coefficients (S), which relate to the rate of
movement, or sedimentation, of a molecule in a centrifugal field. Three general types of RNA are present in cells:
1. Ribosomal RNA (rRNA) is a structural and catalytic RNA, which together
with about 85 different proteins, forms the ribosomes. It is the most abundant RNA in the cell, comprising about 75% of total cellular RNA. In eukaryotes, rRNA exists in four forms: 18S RNA, 28S RNA, 5.8S RNA and 5S RNA.
2. Transfer RNA (tRNA) and small nuclear RNAs are small, stable RNAs.
tRNA consists of about 75 nucleotides and is responsible for amino acid
transfer during protein synthesis. Small nuclear RNA (snRNA) has enzymatic activity and is involved in RNA splicing.
3. Messenger RNA (mRNA) is the copy of a gene that codes for a specific
protein. It is initially transcribed as a large precursor molecule, called heterogeneous nuclear RNA (hnRNA). HnRNA is enzymatically cleaved by
snRNA to remove introns and to form mature mRNA. Messenger RNA is
only a small portion of cellular RNA comprising less than 5% of total RNA
in the cell.
Messenger RNA and translation of protein
Transcription of protein-coding genes results in large hnRNA, containing
introns and exons of the gene. The introns are enzymatically removed from
the hnRNA and the remaining exons spliced together to mature mRNA molecule. Messenger RNA of eukaryotic cells undergoes further modifications
that result in several unique characteristics (Fig. 2.5). The 5′ end of the molecule, where translation is initiated, is capped by methylation of the guanosine molecule. This provides protection from phosphatase and 5′
22
Chapter 2
Fig. 2.5. Anatomy of the messenger RNA molecule.
exonuclease degradation. A leader sequence, also called the 5′ untranslated
region (UTR), follows the 5′ end of the molecule. This 30- to 50-nucleotide
stretch is thought to provide proper alignment with the protein synthetic
material. The coding region, consisting of triplet nucleotides, which each
code for a single amino acid, usually has an initial AUG (Met) codon as the
translation initiation sequence.
At the end of the coding region, nonsense codons (UAA, UAG or UGA)
signal the termination of translation. The trailer sequence (also called the 3′
UTR) follows. This consists of 50 to 150 nucleotides and may provide stability to the mRNA. Within this region is the AAUAAA sequence, which codes
for addition of the polyA tail. The 3′ end of the mRNA molecule has a characteristic polyadenylic acid (polyA) sequence of 50 to 250 adenosines. This
occurs in 97% of all mRNA species, although the mRNA which codes for
chromatin protein histone (~30% of all mRNA) is not polyadenylated. The
polyA tail is believed to protect against 3′ exonucleases and may enhance
translational efficiency.
Translation of mRNA into the final protein product occurs in the cell’s
cytoplasm and involves the interaction of mRNA, tRNA and ribosomes. The
information coded in mRNA consists of groups of three bases, called codons,
each specifying a particular amino acid (Table 2.2). These codons are recognized by complementary triplet bases, the anticodon, located on the tRNA
molecule. Transfer RNAs carry specific amino acids specified by the anticodon–codon to the mRNA. Ribosomes are large macromolecular structures
that coordinate the interaction between mRNA and tRNA to synthesize a
protein or polypeptide. These large structures consist of rRNA core and over
85 small, basic, surface proteins surrounding the rRNA. A eukaryotic ribosome has an overall size of 80S and consists of two subunits, a large 60S subunit that contains 28S, 5S and 5.8S rRNA and a smaller 40S subunit that
contains 18S rRNA. Translating the mRNA sequence into a protein chain
takes place in the space between the two ribosomal subunits. The process of
protein translation involves the initiation of the process by binding to the initiation codon (AUG) of the mRNA, followed by chain elongation in the order
specified by the mRNA codon, and finally chain termination at termination
codons (UAA, UAG or UGA), when translation stops, and the ribosomal
subunits dissociate.
Cellular and Molecular Biology
23
Table 2.2. The genetic code.
Second base
First base (5′end)
U
C
A
G
U
Phe
Ser
Tyr
Cys
U
Phe
Ser
Tyr
Cys
C
Leu
Ser
Ter
Ter
A
Leu
Ser
Ter
Trp
G
Leu
Pro
His
Arg
U
Leu
Pro
His
Arg
C
Leu
Pro
Gln
Arg
A
Leu
Pro
Gln
Arg
G
Ile
Thr
Asn
Ser
U
Ile
Thr
Asn
Ser
C
Ile
Thr
Lys
Arg
A
Met
Thr
Lys
Arg
G
Val
Ala
Asp
Gly
U
Val
Ala
Asp
Gly
C
Val
Ala
Glu
Gly
A
Val
Ala
Glu
Gly
G
C
A
G
Third base (3′end)
RNA polymerases
Three types of RNA polymerase enzymes catalyse the transcription of RNA
from DNA. RNA polymerase I transcribes tandem repeat ribosomal RNA
genes located in the nucleolus to produce 18S, 28S and 5.8S RNAs, structural
components of rRNA. RNA polymerase II transcribes all protein-coding
genes to form mRNA. In addition, RNA polymerase II catalyses the formation of most snRNAs involved in RNA splicing. The formation of tRNA and
5S RNA is catalysed by RNA polymerase III.
Transcription of genes by RNA polymerases I and III is relatively
straightforward. These polymerases catalyse the formation of large quantities of rRNA and tRNA, respectively. These are relatively homogeneous
24
Chapter 2
species of RNA that are not translated into proteins. There is only a single
promoter for the transcription of rRNA genes by RNA polymerase I and
transcription of tRNA genes is regulated by two promoters.
RNA polymerase II, on the other hand, is responsible for catalysing the
transcription of thousands of different genes, each coding for different proteins in the cell. A single cell may contain 10,000 to 20,000 different mRNAs,
each present in widely different concentrations, which vary by tissue and
during different developmental and metabolic stages. Thus, the differential
and specific transcription of multiple, unique, genes by RNA polymerase II
is complex. This process is highly regulated by interactions of RNA polymerase with specific nuclear proteins that assist in assuring that genes are
expressed appropriately.
Regulation of gene transcription
Compared to prokaryotes, eukaryotic genes are relatively complex.
Eukaryotic genes consist of the gene itself and several regulatory gene elements that flank the protein-coding part of the gene. All cells of a plant or
animal contain exactly the same genes, but cells use only a small subset of
these genes. The control of gene transcription is initiated in the promoter
region of the gene, located upstream (5′) to the gene itself. The promoter is
defined as the DNA sequences needed to initiate gene transcription. The
promoter region binds RNA polymerase and other regulatory proteins necessary to initiate transcription. The regulation of gene expression is under
control of regulatory proteins that bind to regulatory elements in the promoter and activate or inhibit gene transcription. These proteins are called
transcription factors and they bind to other proteins and to specific
sequences in the promoter region of the gene called control elements. When
a transcription factor inhibits transcription, it is called a repressor and if a transcription factor stimulates transcription it is called an inducer or activator.
Genes can be classified as those that are always active, producing gene
products at a basal state, and those in which transcription is regulated.
Genes that are constantly transcribed are those that code for structural
proteins of the cell and those involved with cell metabolism. These basal,
constitutively active genes are called housekeeping genes. On the other
hand, regulated genes are activated or repressed only during specific metabolic processes. These would include genes regulated during cell growth
and differentiation, or in response to specific stimuli, such as hormone activation or cell stress. The focus here is on regulated genes that respond to
changes in the cell environment.
The promoter region of most genes that code for proteins contains a
DNA sequence called the TATA box, with the sequence TATAA. This
sequence is located at the 3′ end of the promoter region, about 30 bases
upstream from the transcription initiation site of many genes. This is the element that binds RNA polymerase II. However, polymerase II by itself cannot
initiate transcription. A basal level of transcription requires the presence of
Cellular and Molecular Biology
25
several transcription factors along with RNA polymerase II. These are called
general transcription factors (GTF) and over 20 of them have been discovered.
The initiation of gene transcription requires the ordered, sequential assembly of six of these GTFs into a macromolecular aggregate with RNA polymerase II. The first of these GTFs is called the TATA box-binding protein
(TBP). This factor is common to all three polymerases and can help to initiate transcription in genes regulated by RNA polymerases I, II and III. The
initial assembly of the transcription complex for genes transcribed by RNA
polymerase II involves the binding of TBP to other proteins to form the transcription factor TFIID, which binds to the TATA box and opens the doublestranded DNA. Binding of other factors TFIIA and TFIIB provides stability
to the complex and assures the selection of the correct transcription start site.
It is only now that RNA polymerase II, bound to another transcription factor
(TFIIF), binds to the TATA box. The final GTFs involved in the initiation of
basal transcription, TFIIE and TFIIH, provide the final components of the
transcription apparatus (Fig. 2.6). From this brief description, it is apparent
that initiation of basal transcription involves the cooperation of a variety of
regulatory proteins to provide the efficient transcription of a gene.
Transcriptional control of specific genes
Control of the transcription of specific genes involves additional regulatory
elements. While the general transcriptional elements and associated proteins
can induce limited expression of a gene, the full activation of gene expression requires other promoter elements. There are two types of elements:
proximal promoter elements (PPE) and enhancer elements. The PPE are
located near the gene, 50 to 250 bp upstream from the transcriptional start
site. These are small stretches of DNA (<10 bp) that bind additional regulatory proteins. Two important examples of PPEs are the CAT box, with the
sequence CCAAT, and the SP1 site, with the sequence GGCGCC. Both sites
are present in many housekeeping genes and are required for full transcriptional activity. Most promoters have many different PPEs, also called
response elements, which respond to a variety of factors, ranging from metals to transcription factors that mediate the effects of hormones. Thus, specific gene transcription is regulated by the presence of several specific
Fig. 2.6. The eukaryotic transcription complex.
26
Chapter 2
response elements associated with a particular gene. The effectiveness of
these response elements depends upon the relative availability and activity
of multiple protein transcription factors. The presence of PPEs and their
associated transcription factors adds another level of regulatory control and
complexity to control the expression of genes. This complex array of regulatory elements and their associated transcription factors assures a fine-tuned
response to factors that alter gene expression.
The enhancer elements are another type of promoter element that controls
gene expression. Enhancer elements are characterized by their ability to
affect gene transcription when located at great distances, up to 10 kb, from
the gene. Enhancer elements may also be present within the genes themselves, within the introns of genes. They participate in the regulation of
many, if not all genes. They are able to regulate gene transcription from great
distances as the regulatory proteins that bind to them also bind to other proteins attached to distant sites on DNA. These protein–protein interactions
are possible because of loops in the DNA strand. This allows the physical
contact of proteins bound to distant enhancer elements with those near the
transcription start site. As their name implies, enhancer sequences interact
with transcription factors and enhance the rate of specific gene transcription,
although they cannot initiate transcription by themselves. Enhancer elements for tissue-specific transcription factors confer transcriptional regulation on a tissue-specific basis.
Transcription factors
Hundreds of gene-specific transcription factors have been discovered since
the 1990s. It is estimated that approximately 6% of the human genome
(total DNA) is devoted to the production of these proteins. Despite this
abundance, the regulation of specific gene transcription involves not a single unique transcription factor, but requires different combinations, specific
to each gene, of several of these proteins. Although most of these transcription factors are activators of gene transcription, repressors have also
been identified.
Transcription factors are classified based upon the structure of their
specific DNA-binding domains. There are three basic types:
1. Homeodomain proteins contain a conserved 60 amino acid domain (the
homeobox) and a helix–turn–helix domain. Both domains interact with specific DNA sequences. There are more than 150 of these transcription factors
in humans. Many homeodomain proteins play important roles in the development of embryonic structures such as the muscles, limbs and nervous
system.
2. Zinc finger proteins contain multiple polypeptide ‘fingers’ consisting conserved pairs of cysteines and histidines that bind a single zinc ion. More than
600 of these proteins exist in humans. The tip of the zinc finger interacts with
bases in DNA. Steroid nuclear receptors, which contain a specific ligand-
Cellular and Molecular Biology
27
binding domain for steroid attachment and a DNA-binding domain, are in
this category of transcription factors.
3. Leucine zipper proteins contain a basic amino acid region that binds to DNA
and a series of repeated leucine residues. The leucine repeats form an α-helix
that binds other leucine zipper molecules to form a dimer. An important regulatory transcription factor that initiates adipose tissue formation, called
CAAT/enhancer-binding protein (C/EBP), is an example of a leucine zipper
protein. Basic helix–loop–helix (bHLH) proteins are similar to leucine zipper
proteins but have two dimerization domains separated by a loop region. The
muscle regulatory factors (MRFs), such as MyoD, that induce muscle differentiation are examples of bHLH transcription factors.
Biotechnology and Genetic Engineering
Biotechnology is defined as the use of technologies based upon living systems to develop commercial processes and products – making a buck from
biology. Some examples of biotechnology directed toward animal production systems include gene transfer to produce transgenic animals that contain a foreign gene that may enhance growth or the production of animal
products such as meat, milk or fibre. The processes of embryo manipulation
and tissue culture are also considered as biotechnological processes.
Biotechnology is used for bioprocess engineering, in which production of
specific animal proteins using bacterial or mammalian cell cultures is accomplished. The process of producing monoclonal antibodies is a large and
important field of biotechnology. Monoclonal antibodies are produced from
immortalized clones of antibody-producing lymphocytes. As such, these
antibodies are very specific for their antigen targets and are used for the
diagnosis and prevention of many diseases.
The relatively recent explosion in the use of biotechnology after the
1980s has come about because of discoveries in the chemistry and manipulation of genes at the molecular level. The production of complex proteins
containing hundreds of amino acids in the proper sequence cannot be done
economically or efficiently using synthetic organic chemistry methods.
Instead, a living system, bacterial or eukaryotic, must be involved in the
sequential ordering of amino acids to form a biologically active, functional
protein. For this purpose, recombinant DNA is produced. A simple definition of recombinant DNA is that DNA from one source recombined with
DNA from another, often bacterial or viral, source.
Introduction of a specific gene that codes for a particular protein has
many uses. After introduction of a gene into a bacterium, the bacteria can be
grown en masse to amplify the foreign gene. That gene can then be used in
the production of specific proteins, either in vivo or in vitro. After removal of
the amplified foreign gene from the bacteria, it can be used to produce transgenic plants or animals with the goal of improving agricultural production.
About 70% of the soybeans and cotton that are grown in the USA are now
28
Chapter 2
derived from transgenic organisms, as is 30% of the US maize crop. Many of
the transgenic plants are engineered to be resistant to the glyphosate herbicide Roundup® or to contain the naturally occurring insecticide Bacillus
thuringiensis toxin gene for resistance to insects. A more ambitious goal for
human medicine is to use specific genes to correct diseases of genetic origin.
Transgenic Animals and their Potential
Only since the mid-1970s have we been able to isolate specific genes,
sequence them and recombine them with the DNA of foreign hosts. The
importance of the ability to make specific proteins cannot be overemphasized.
Knowing the genetic code for amino acids and proteins allows them to be
modified – ‘nature improved upon’ amino acid sequences can be changed to
reduce degradation (increase half-life) of proteins, increase the efficiency of
enzymes, or to increase hormone/receptor efficiency.
In the context of animal agriculture, animals produced with an added
gene can be used to improve productivity (produce more meat, fibre or milk
with less or the same nutrient intake), as measured by growth rate, feed efficiency, milk efficiency, wool production or reproductive efficiency. An example of the latter is the potential to increase twin lambs in sheep by
introducing the Booroola gene for twinning. Insertion of antibody genes to
enhance immune function to a specific disease might be used to induce disease
resistance of animals. In addition, transgenic animals that express a specific
gene into an easily accessible compartment, such as milk, allows the animal
to be used as a ‘bioreactor’, capable of producing a specific protein. This has
the advantage of high-yield productions in a mammalian host that would
lack possible bacterial toxins that may be present in recombinant products
isolated from bacterial cultures. Introduction of specific genes, for example
the gene for GH or leptin, has the potential to alter body composition by
increasing lean body deposition with a reduction in body fat. Alteration of
the ruminant microbial genome has the potential to alter the efficiency of
rumenal digestion. Transgenic animals provide the opportunity for ‘instant
evolution’, as the introduced gene or genes are incorporated into all cells of
the body, including the germ line cells. Thus, dramatic, heritable changes
may be introduced into an animal and, if successful, could save generations
of breeding and selection for favourable traits.
Identifying, Isolating and Transferring Genes
The mammalian genome has about 3 to 4 billion bp of DNA. Of this number,
there are about 35,000 to 50,000 genes that code for protein products. As a
single gene consists of 1000 to 5000 bases, this accounts for less than 10% of
the total genomic DNA. Most estimates suggest that ~95% of genomic DNA
does not code for proteins. The majority of DNA is believed to be regulatory
in nature, assuring that genes are expressed at the proper time in develop-
Cellular and Molecular Biology
29
ment, by a specific cell and in response to a specific environmental cue.
When cellular RNA is considered, only a small proportion of the total RNA
is devoted to mRNA that codes for proteins. Roughly 2% to 5% of cellular
RNA is mRNA, while the rest is transfer and ribosomal RNA. With this
knowledge, it is apparent that the isolation of a single specific gene from this
tangle of DNA is a Herculean task.
Genes can be isolated in several different ways. Chemical synthesis (if
sequence is known) can produce only relatively small pieces of DNA (a few
hundred bases, with luck). Another approach is to digest total DNA, using
restriction endonucleases (RE), into 15–20 kb fragments. The digested DNA
is inserted into plasmids or bacteriophages and grown in the host to amplify
the DNA fragments. This is called a genomic library, which consists of thousands of pieces of DNA, mostly non-coding introns and repetitive DNA.
This approach is used primarily for DNA sequencing of entire genomes.
The most common approach to the isolation of the gene that codes for a
specific protein is to prepare a complementary (cDNA) library.
Complementary DNA is a copy of the mRNA present in a specific tissue or
cell at a specific time. After isolation of total RNA from the organ or tissue of
interest, the mRNA fraction is isolated by binding to a synthetic polyT resin,
either in an aqueous column or in a test tube. As the majority of mRNA has
a polyA sequence that is complementary to the polyT sequence, mRNA will
be selectively enriched from the total RNA, thereby eliminating 95% to 99%
of the total RNA. Messenger RNA is then subjected to reverse transcription
to prepare cDNA from total mRNA. The cDNA is then introduced into bacterial plasmids (or bacteriophages) and amplified by growing the recombinant organisms in vitro. This is called a cDNA library. It consists of cDNA
derived only from the mRNA population of the cell and only the exons of
the gene are present. After reintroduction of the recombinant plasmids into
the bacteria, the bacteria are allowed to grow, reproducing and amplifying
the plasmids. Bacterial colonies containing the genes of interest are then
identified and selected with probes that contain sequences complementary
to the mRNA of interest. After amplification of the foreign DNA of interest,
it is then removed from the plasmid using restriction enzymes, purified and
used in an animal (or plant) host to produce a transgenic organism. This
process is outlined below.
The use of bacterial RE has allowed the isolation, manipulation and
recombination of DNA. RE, also called restriction enzymes, are bacteria’s
natural defence system that protect the bacterium from viral (bacteriophage)
invasion. They protect bacteria by digesting foreign, non-methylated viral
DNA, while methylation of bacterial DNA protects it from self-digestion.
They are called RE because they restrict the growth of viral bacteriophages
in bacterial hosts. Restriction enzymes bind to and cleave DNA at specific
sequences. Due to the specific site of ‘cutting’ and reproducibility of these
enzymatic reactions, production of DNA fragments can be controlled and
the generation of specific sequences of DNA can be reproducibly performed.
Restriction enzymes are given names that are abbreviations of the bacteria
from which they came (Fig. 2.7). Restriction enzymes cut double-stranded
30
Chapter 2
Fig. 2.7. Restriction endonucleases and their sites of action.
DNA at areas with a twofold axis of symmetry. These palindromic sequences
have the same sequence from left to right as they do in the reverse order.
Examples of language palindromes include, e.g. ‘Madam I’m Adam’; ‘A man,
a plan, a canal, Panama’. Cleavage of double-stranded DNA by restriction
enzymes may leave blunt ends, in which the two strands of DNA are cut at the
same length, with no extended strands. Other restriction enzymes leave staggered, overlapping ends of DNA on their digested strands. These singlestranded extensions of the DNA molecule are called ‘sticky ends’ as they are
available for complementary (A–T or G–C) hydrogen bonding to form doublestranded DNA (Fig. 2.8). Blunt ends can be enzymatically ‘tailed’ to introduce
restriction enzyme sites and/or sticky ends available for hydrogen bonding.
Fig. 2.8. Production of blunt or sticky ends of DNA by restriction endonucleases.
Cellular and Molecular Biology
31
The bacterial chromosome consists of a single circular molecule of
‘naked’ DNA. In addition, bacteria have small (103 to 105 bp) circular, auxiliary DNA called plasmids. Plasmids are abundant (20–30+ copies per cell),
self-replicating, and are transferred from one bacterium to another. Plasmids
are used as the insertion site for the introduction of foreign DNA into bacteria to create recombinant DNA. Bacteriophages, viruses that infect bacteria
by injecting their DNA into them, may also be used to introduce foreign
DNA into bacteria. Some plasmids carry genes for resistance to antibiotics
such as ampicillin or tetracycline. When foreign genes are inserted into the
plasmid’s antibiotic resistance genes, the resistance genes are disrupted,
inducing specific antibiotic susceptibility. This provides a simple method to
identify bacteria containing foreign DNA, as the recombinant bacteria,
grown in duplicate cultures, are killed by exposure to specific antibiotics.
Synthetic plasmids are now widely available commercially which contain
inserted RE sites and antibiotic resistance genes. An example of this is the
pBR322 plasmid, which contains both ampicillin and tetracycline resistance
genes and DNA sequences for specific restriction enzymes (Fig. 2.9). Newer
synthetic plasmids are equipped with colour-producing genes for easy identification of foreign gene insertion, so that selection for recombinant clones
is simplified.
To make recombinant DNA, both plasmid DNA and isolated foreign
cDNA are digested with the same RE, to produce complementary sticky
ends. The BamHI enzyme can be used for this as a restriction site for this
enzyme disrupts the tetracycline resistance gene of the pBR322 gene
(Fig. 2.10). After plasmid and foreign gene DNA are digested with BamHI, they
are combined and heated to 70˚C to separate the hydrogen-bonded strands.
The reaction mixture is then cooled to allow the complementary sticky ends
Fig. 2.9. The pBR322 plasmid. Some restriction endonucleases sites (arrows) are shown. Amp,
ampicillin resistance gene; Tet, tetracycline resistance gene; Ori, replication origin site.
32
Chapter 2
Fig. 2.10. Introduction of foreign DNA into the pBR322 plasmid.
to recombine. When the treatment is complete, unwanted by-products are
also formed in addition to the desired plasmid–gene recombinant DNA.
These include intact plasmids and intact genes that must be removed from
the recombinant DNA. The recombinant DNA at this stage is attached only
by non-covalent hydrogen bonds between the complementary bases of the
DNA strands of the sticky ends. To covalently incorporate the foreign DNA
within the plasmid, the enzyme T4 ligase is used. The plasmids are reintroduced into bacteria, using either osmotic shock – high calcium (20–50 mM),
or electrophoretic transfer (electrophoration) and plated on to an agar
growth medium. Bacteria are allowed to grow into visible colonies. The
colonies that contain the recombinant DNA are selected using replicate plating – colonies picked up with a felt template and transferred to antibioticcontaining agar – recombinant colonies are identified by their sensitivity to
antibiotics, in this case tetracycline, and selected for clonal growth.
Alternatively, colony replicates on nitrocellulose paper can be hybridized to
complementary, labelled probes specific for the inserted DNA to identify
positive colonies. Newer technology uses synthetic plasmids, e.g. pGEM,
which contain genes (LacZ = β-galactosidase) that can be detected by colour
screening with appropriate substrate. Intact LacZ produces blue colonies.
Cellular and Molecular Biology
33
Positive colonies are then grown in large suspension cultures, plasmids isolated, digested with the restriction enzyme used to produce the original
recombinants and the amplified foreign gene is purified.
To introduce the amplified gene into a multicellular animal, it is microinjected into a newly fertilized oocyte, transferred into a foster mother and
allowed to come to term. This results in random incorporation of the gene
into host chromosomes. There are several problems associated with random
incorporation of DNA into the host chromosomes. The success rate is low,
~1% viable transgenics are produced from the injected eggs. The chances of
introducing a lethal mutation are high, as the foreign gene may be incorporated into an existing gene that is essential for metabolism. When genes are
randomly incorporated into the oocyte, all body cells, somatic and germ line
cells, contain and express the gene. When the gene is introduced without
appropriate regulatory controls, inappropriate developmental gene expression occurs and the introduced gene is expressed in embryonic, fetal, neonatal and adult animals. The normal developmental, tissue-specific regulation
of gene expression is lost and the gene is either fully expressed or not.
High levels of unregulated gene products lead to pharmacological effects
and metabolic diseases. The hit-or-miss random incorporation of genes leads to
low success rates. These problems have been addressed by targeting genes
to specific sites in the animal’s genome and by adding regulatory elements
that restrict gene expression to specific organs during specific developmental stages and in response to specific regulatory signals.
Homologous recombination, gene targeting and gene knockouts
When a foreign gene is introduced into mammalian cells, it is incorporated
into DNA at random locations. Most of the time the foreign gene recombines
with the host DNA at a site that is unrelated to the introduced gene. This is
called heterologous recombination. In a very few instances, about 1 in 1000,
the gene recombines with the identical site of the original gene by a process
called homologous recombination. Methods to increase rates of homologous
recombination of introduced genes or to enrich cell populations that have
undergone homologous recombination offer powerful tools to study gene
function and to produce transgenic animals that are more physiologically
viable. Homologous recombination can be used to target gene insertions to
an exact known location in the genome. This provides the potential for gene
replacement, in which a functional gene can be used to replace a defective
one. In animal production, one can envision the replacement of a naturally
occurring gene with one that is genetically engineered, to produce a gene that
is more efficiently regulated or one that produces a protein product with
enhanced biological activity.
At present, homologous recombination is used primarily to disrupt specific genes in an animal. Animals that have successfully undergone homologous recombination can be produced. Mice are used for this process owing
to their abundance, well-known genetics and short generation times.
34
Chapter 2
Animals in which the gene has been disrupted are termed gene knockout
mice. This method has provided a powerful tool to study the functions of
specific gene products. The gene to be disrupted is modified in vitro by the
insertion of a selectable marker gene that has no promoter (Fig. 2.11). The
marker gene confers drug resistance on the cell and its presence protects the
cell from lethal drug effects. A marker commonly used in mammalian cells
is a bacterial gene that confers resistance to a neomycin-related drug, G418.
This drug kills mammalian cells by inhibiting protein synthesis. The gene is
transfected into embryonic stem cells maintained in vitro. Embryonic stem
cells are undifferentiated pluripotent cells derived from the inner cell mass
of the embryonic blastocyst. They are used because of their ability to differentiate into all cell lineages of the animal, including the germ cells that will
form spermatozoa and ova. When the promoterless gene is incorporated into
its appropriate locus in the genome, the endogenous promoter activates it
and the gene product coding for G418 resistance is expressed. Cells without
the drug resistance gene die upon exposure to G418 while those that have
incorporated the gene via homologous recombination survive. These cells
are then isolated, cloned and injected into a blastocyst, which is transplanted
into a foster mother and allowed to come to term (Fig. 2.12). Many of the
resulting pups are chimeras, containing wild-type cells from the blastocyst
and the introduced stem cells. These chimeric animals can be easily identified if one uses embryonic stem cells derived from black mice and donor
embryos collected from homozygous white mice, or vice versa. Chimeric
animals, containing the modified gene, are characterized by black and white
patches of fur. These mice can be screened with probes complementary to
the gene of interest to ensure the presence of the appropriate targeted allele
and then interbred with animals homozygous for coat colour to produce
animals homozygous for the desired mutation.
This method for gene disruption has been refined in recent years to eliminate some of the problems associated with traditional gene-targeting meth-
G418 resistance gene
Cloned gene X with promoterless
G418 resistance gene
No promoter
Non-homologous recombination
G418 resistance not expressed
cell is G418 sensitive
Fig. 2.11. Homologous recombination and gene disruption.
Gene X promoter
Homologous recombination
G418 resistance expressed
gene X is disrupted
cell is G418 resistant
Cellular and Molecular Biology
35
Fig. 2.12. Production of mice with knockout genes.
ods. As with transgenic animals, the disrupted gene is present in all tissues at
all stages of development. This can lead to relatively non-specific effects and
to embryonic mortality. Tissue-specific or developmentally regulated genes
36
Chapter 2
can be disrupted using the Cre-lox recombinase system. Recognition sites on
DNA for the Cre-recombinase enzyme, called loxP sites, are inserted into both
ends of the gene to be disrupted and transgenic mice with the insertion are
produced by injecting the modified gene into embryos. Another set of mice is
generated with the Cre-recombinase enzyme linked to tissue-specific promoters. Mating of mice engineered with the flanking loxP sites with those producing the Cre-recombinase enzyme produces animals in which a specific
gene is disrupted in a developmental or tissue-specific manner.
Animals produced by these methods have provided valuable tools to
study the functional roles of hormones and growth factors. Elimination of
specific hormone, growth factor or transcription factor genes produces animals with altered metabolic or developmental traits. Many times, these
mutations are lethal and animals die before embryonic and fetal development is complete. In other cases, disruption of specific genes produces a
viable animal that is stunted or an animal that is deficient in a specific tissue
such as bone, fat or muscle. Knockout mice and their uses will be addressed
in subsequent chapters that deal with the development of specific tissues,
growth factors and transcription factors.
References and Further Reading
Betz, U.A., Vosshenrich, C.A., Rajewsky, K.
and Muller, W. (1996) Bypass of lethality
with mosaic mice generated by CreloxP-mediated recombination. Current
Biology 6, 1307–1316.
Capecchi, M.R. (1989) Altering the genome
by homologous recombination. Science
244, 1288–1292.
Hawkins, J.D. (1985) Gene Structure and
Expression. Cambridge University Press,
Cambridge, UK, 173 pp.
Pollard, T.D. and Earnshaw, W.C. (2002)
Cell Biology. W.B. Saunders, Elsevier
Science, Philadelphia, Pennsylvania,
805 pp.
Novikoff, A.B. and Holtzman, E. (1970) Cells
and Organelles. Holt, Rinehart and
Winston, New York, 337 pp.
Watson, J.D., Gilman, M., Witkowski, J. and
Zoller, M. (1992) Recombinant DNA,
2nd edn. W.H. Freeman, New York,
626 pp.
3
The Endocrine System
The endocrine system is an essential regulatory component of animal physiology, metabolism and growth. The endocrine system plays a central role in
the regulation of nutritional intake and utilization, and integrates the multiple functions of physiological systems. Unlike enzymes, which are secreted
into the gastrointestinal tract by glands in the tract, hormones are secreted
directly into the bloodstream by a variety of ductless glands located
throughout the body.
Starling, in 1905, defined a hormone as a ‘chemical messenger which is
speeding from cell to cell along the bloodstream, may coordinate the activities and growth of different parts of the body’. Stated differently, we now
know that a hormone is a chemical messenger secreted by a discrete, ductless gland directly into the bloodstream, which carries it to its distant, usually well-defined target cells that contain specific hormone receptors, where
the hormone elicits a response. Hormones circulate and are active at very
low concentrations (10−9 to 10−12 M). More recently, the definition of a hormone has been broadened, as some hormones that were thought to be confined to a single source are now known to be produced by multiple tissues.
For example, the products of the pancreatic islets, the pituitary and thyroid
glands were thought to be strictly defined hormones. In the past few years,
it has been shown that insulin, glucagon and thyroid hormones are produced at sites distant from their respective glands. These locally produced
hormones may stimulate changes in the growth and metabolism of cells
without the intervention of the circulatory system. Other examples of hormones, which also act as local growth factors, are the IGFs, leptin and somatostatin (SS). Hormones and growth factors can act in multiple ways, which do
not involve the intervention of the circulatory system (Fig. 3.1). Thus,
autocrine hormones are those which affect the same cell that produced them,
while paracrine hormones are produced in one cell type and affect an adjacent cell type. Neurohormones are similar to traditional hormones but differ
only in their site of synthesis. Thus, neurohormones are produced in neural
tissues and secreted into the bloodstream. Examples of these hormones are
oxytocin and vasopressin, produced by the posterior pituitary gland, an
extension of the CNS, and released into the bloodstream. In addition, neurohormones are produced by the hypothalamus at the base of the brain and are
©K.L. Hossner 2005. Hormonal Regulation of Farm Animal Growth (K.L. Hossner)
37
38
Chapter 3
Fig. 3.1. Endocrine, paracrine and autocrine mechanisms of hormone delivery.
carried by a portal system to the anterior pituitary gland. These local, specialized hormones affect the anterior pituitary gland, inducing the stimulation or inhibition of anterior pituitary hormone secretion. Neurohormones
must be distinguished from neurotransmitters, such as acetylcholine, serotonin and γ-aminobutyric acid (GABA), which provide communication
across neural synapses. Neurotransmitters allow nerves to communicate
with each other and with muscles.
Hormones regulate such diverse functions as respiration and metabolic
rate, reproduction, cell, tissue and organ growth, glucose and mineral balance, blood pressure and gastric acid secretion. Table 3.1 illustrates the
major endocrine glands and their hormones. This is not an all-inclusive compilation, as the focus is on hormones which are directly involved in growth.
Hormones such as leptin, from adipose tissue, IGFs, from liver and many tissues, and vitamin D3, synthesized by multiple tissues, are not included in
this compilation of glands and hormones. Leptin, IGFs and vitamin D will
be discussed thoroughly in subsequent chapters.
The location of the endocrine glands in the cow is shown in Fig. 3.2. The
pituitary gland in the adult lies at the base of the brain, in a secure bony concavity of the skull called the sella turcica. The pituitary gland is attached to
the brain and has three distinct portions, each synthesizing and secreting
distinct hormones with far-ranging effects on growth and metabolism.
Embryonically, the anterior pituitary gland (also called the pars distalis
The Endocrine System
39
Table 3.1. The endocrine glands and their hormones.
Gland
Hormones
Major actions
Anterior pituitary
(pars distalis)
Growth hormone (GH)
and somatotropin (ST)
Muscle/bone growth; protein
synthesis; lipid and carbohydrate
metabolism
Adrenocorticotrophin (ACTH)
Controls adrenal cortex secretion
of cortisol, corticosterone
Thyrotrophin (thyroid-stimulating
hormone (TSH))
Stimulates synthesis and release
of thyroid hormones from thyroid
Leutinizing hormone (LH) and
follicle-stimulating hormone (FSH)
Regulate steroid synthesis and
release from the gonads
Melanocyte-stimulating
hormone (MSH)
Melanin synthesis in skin;
skin darkening; appetite effects
Intermediate
pituitary (pars
intermedia)
Posterior pituitary Oxytocin
(neurohypophysis)
Milk ejection; uterine contraction
Vasopressin, antidiuretic
hormone (ADH)
Increases blood pressure;
water reabsorption in kidney
Thyroxine (T4),
triiodothyronine (T3)
Growth, neural development,
regulates metabolic rate
Calcitonin (CT)
Lowers blood calcium, phosphate
Parathyroid gland
Parathyroid hormone (PTH)
Increases blood calcium, lowers
blood phosphate
Adrenal cortex
Cortisol, corticosterone
Induce gluconeogenesis, protein
catabolism; anti-inflammatory actions
Adrenal medulla
Epinephrine (adrenalin)
Glycogen breakdown; increases
blood glucose, blood flow, heart rate
Norepinephrine (noradrenalin)
A neurotransmitter; increases
blood pressure
Pancreatic
islets (β-cells)
Insulin
Reduces blood glucose;
increases tissue use of glucose,
synthesis of lipid and protein
Pancreatic
(α-cells)
Glucagon
Increases blood glucose;
catabolism of protein, lipid; reduces
gluconeogenesis
Testes
Testosterone
Male sexual characteristics,
muscle growth
Ovaries,
placenta
Oestrogens, progestagens
(i.e. progesterone)
Female sexual characteristics;
maintenance of pregnancy
Thyroid gland
40
Chapter 3
Fig. 3.2. Location of the endocrine glands in a cow.
or adenohypophysis) is derived from the ectoderm of the pharynx from an
embryonic structure called Rathke’s pouch. The anterior pituitary gland was
once considered ‘the master conductor of the endocrine orchestra’, because
it controls the function of many other endocrine glands such as the thyroid,
adrenal cortex and the gonads. It is now known that each of the anterior
pituitary hormones from the ‘master conductor’ is controlled by neurohormones secreted by the hypothalamus (discussed later) and the pars distalis
might now be considered an ‘assistant maestro’. In addition to the hormones
which regulate other glands, the anterior pituitary produces GH, also
called somatotropin (ST or STH), a hormone that plays an important role in
growth and intermediary metabolism. The intermediate pituitary
(pars intermedia), also a derivative of Rathke’s pouch, lies between the anterior and posterior pituitary glands and secretes melanocyte-stimulating hormone (MSH) that has effects on skin pigmentation. The posterior pituitary,
or neurohypophysis, has a separate embryonic derivation, and is formed by
an evagination of the ventral diencephalon of the embryonic brain. It is a
neural tissue and remains connected to the brain by the infundibular stalk.
The posterior pituitary is the site of synthesis of oxytocin and vasopressin,
neurohormones which regulate milk release and blood pressure.
The thyroid gland is a paired, butterfly-shaped organ which is located just
below the larynx of the trachea. It secretes the hormones thyroxine (T4) and triiodothyronine (T3) which are essential for normal development of the CNS and
play important roles in the regulation of metabolic rate and oxygen consumption by most cells of the body. The thyroid hormones are examples of hormones
that are modified amino acids. As shown in Fig. 3.3, they are synthesized by iodination of tyrosine to form monoiodotyrosine and diiodotyrosine. These are
then combined to form the active T3 and T4 thyroid hormones. The thyroid
gland also secretes calcitonin (CT), a product of the C-cells of the thyroid.
CT plays an important role in the growth and remodelling of bones,
The Endocrine System
41
Fig. 3.3. Synthesis of the thyroid hormones from tyrosine.
reflected in its effects on lowering blood calcium. The parathyroid glands exist
as two paired glands embedded in the back of the thyroid glands and have
effects on blood and bone calcium, which are opposite to those of CT.
The adrenal glands are paired endocrine glands which reside on the anterior, or cephalic, portion of the kidneys. Grossly, the two functional areas of the
adrenal glands are the outer cortex, which produces the corticosteroid hormones
cortisol and corticosterone, and the inner medulla, which produces the catecholamines epinephrine (adrenaline) and norepinephrine (noradrenaline).
Epinephrine is the primary circulating hormone produced by the adrenal
medulla, while norepinephrine, which circulates in lower concentrations, acts
primarily as a neurotransmitter. Adrenal hormones are released primarily in
42
Chapter 3
response to stress, and stimulate the mobilization of carbohydrates for use as
energy by the body and by increasing heart rate, blood pressure and blood flow
to skeletal muscle. These are the hormones which regulate the ‘fight or flight’
response, enabling an attacked animal to respond to a physical challenge with
an appropriate response, either standing its ground and fighting or running
away from danger. They also have important functions in the maintenance of
daily physiological functions, such as providing a balance to the glucose-lowering effects of insulin. New approaches to the artificial regulation of body composition of meat animals utilize synthetic catecholamines (β-agonists), which
take advantage of the lipolytic effects of these compounds to reduce carcass fat
while increasing lean meat. These are discussed in detail in Chapter 10.
The pancreas produces the important glucose regulatory hormones
insulin (from the β-cells of pancreatic islets) and glucagon, a product of pancreatic α-cells. Insulin is the hormone primarily responsible for stimulating
the uptake and metabolism of glucose by most cells of the body, especially
skeletal muscle and adipose tissue. Insulin is released in direct response to
the increased blood glucose seen after a meal in non-ruminants. Levels of
insulin in ruminants are less variable, as the rumen storage and release of
energy sources (primarily volatile fatty acids (VFA)) is much slower and
blood concentrations of glucose are not as variable as those seen in nonruminants. The overall effect of insulin is to reduce blood glucose levels. The
other major pancreatic hormone, glucagon, acts as an insulin counter-regulatory hormone, mobilizing glucose from hepatic stores and increasing
blood glucose. Other hormones, which also have insulin counter-regulatory
effects on blood glucose, include the corticosteroids and GH. All of these
hormones act to mobilize glucose in one way or another with a resultant
increase in blood glucose and a maintenance of blood glucose levels within
a very narrow concentration range (4–7 mM in humans).
The gonads produce the sex-specific hormones oestrogen in females and
testosterone in males. While these gonadal steroids play a natural role in the
maintenance of sexual characteristics in their respective sexes in all animals,
in ruminants they are also used as growth promotants, which increase feed
efficiency and alter body composition. These are discussed in detail in
Chapter 9.
In addition to classifying hormones based upon their source and function, they can also be described based upon chemical type or composition.
This is important when the synthesis, use, mechanism of action and metabolism of different hormones are considered. There are three general chemical
types of hormones.
1. Protein/polypeptide hormones consist of strings of amino acids. The pituitary
gland produces only protein hormones, such as GH, adrenocorticotrophin
(ACTH) and thyroid-stimulating hormone (TSH). Other glands, such as the
pancreas, also produce relatively small polypeptide hormones such as insulin.
2. Some hormones are relatively simple amino acid derivatives, such as the
thyroid hormones, which are derived from tyrosine, and the adrenal catecholamines epinephrine, norepinephrine and dopamine, also tyrosine
derivatives.
The Endocrine System
43
3. Steroids are non-polar, lipid-soluble cholesterol derivatives. They are produced in the gonads, the adrenal glands and the placenta. Examples of these
complex molecules include the sex steroids, oestrogens, progesterone and
testosterone, and the adrenal steroids, cortisol and corticosterone.
Control of Pituitary Hormone Secretion
The release of hormones from the anterior pituitary is controlled by the
hypothalamus, a specialized area of the midventral brain that lies beneath
the third ventricle and thalamus of the brain and above the pituitary gland
(Fig. 3.4). The hypothalamus consists of specialized neuronal cell bodies that
synthesize and secrete releasing and inhibiting neurohormones into a capillary system called the hypophyseal portal system. This tiny specialized vascular system, which begins in a capillary bed in the hypothalamus and
terminates in a capillary bed in the anterior pituitary, carries neurohormones
from the hypothalamus to the anterior pituitary gland. The neuronal cell
bodies in the hypothalamus are organized into specific areas called hypothalamic nuclei (Fig. 3.5). Each nucleus produces different releasing or
inhibiting neurohormones that travel down axons to be released into the
Fig. 3.4. Anatomy of the pituitary gland.
44
Chapter 3
Fig. 3.5. Location of the hypothalamic nuclei. Abbreviations: AHA, anterior hypothalamic area;
AR, arcuate nucleus; DMN, dorsomedial nucleus; MB, mammillary body; ME, median
eminence; OC, optic chiasma; PHN, posterior hypothalamic nucleus; POA, preoptic area;
PVN, paraventricular nucleus; SC, suprachiasmatic nucleus; SO, supraoptic nucleus;
VMN, ventromedial nucleus.
hypophyseal portal system. For the most part, these are polypeptides containing anywhere from 10 to 43 amino acids. These neurohormones, also
called hypophysiotropic hormones, stimulate or inhibit anterior pituitary
hormone release (Table 3.2). The release of GH is under both positive and
negative hypothalamic control. Growth hormone releasing hormone
(GHRH) is a 43-amino acid polypeptide that stimulates GH release from
Table 3.2. Hypothalamic control of anterior pituitary hormone release.
Hormone
Hypothalamic neurohormones
GH
GHRH (+) and GHRIH (somatostatin) (−)
TSH
TRH (tripeptide)
LH/FSH
GnRH
PRL
Dopamine (−)
ACTH
CRF
The Endocrine System
45
the anterior pituitary, while growth hormone release-inhibiting hormone
(GHRIH), or SS, a 14-amino acid polypeptide, inhibits GH release.
Gonadotropin-releasing hormone (GnRH) is a 10-amino acid polypeptide that
stimulates LH and FSH release, while thyrotropin-releasing hormone (TRH),
a tripeptide, stimulates TSH release. The control of prolactin (PRL) secretion
is under negative control by the neurotransmitter dopamine, a catecholamine derivative. The 41-amino acid corticotrophin-releasing hormone
(CRH) stimulates ACTH release. The posterior, or neural, lobe of the pituitary is an extension of the CNS. Neural cell bodies in the hypothalamus
synthesize the posterior pituitary hormones oxytocin and vasopressin. The
axons of these neurones terminate in the posterior pituitary where they
release these neurohormones into the systemic circulation.
Metabolic hormones such as insulin, GH and the adrenal catecholamines
are involved in glucose, lipid and amino acid homeostasis. These hormones
are controlled by nutritional intake and by systemic levels of metabolites,
primarily glucose. Elevated levels of glucose also act on the hypothalamus
and pituitary to reduce GH secretion while stimulating the release of insulin
from the pancreas.
An essential aspect of the regulation of hormone concentrations involves a
concept called ‘negative feedback control’. This involves the self-regulation of
hormone concentrations (Fig. 3.6). Thus, when blood hormone levels increase,
this increase is detected by specific hormone receptors in the hypothalamus or
the endocrine gland. When a hormone affects the gland that secretes it, this is
called a short-loop negative feedback. A long-loop negative feedback arc is one
that affects the control system preceding the gland of secretion. In this case, the
hypothalamus, not the pituitary gland, would be affected. In response, the secretion of stimulatory neurohormones from the hypothalamus or the hormones
themselves is reduced. This results in a reduction of hormone release from the
gland and lower circulating levels of the hormone. These fine-tuned controls
that regulate hormone concentrations ensure that hormone concentrations do
not exceed a set point of concentration that would harm the animal.
As we have seen, the secretion of hormones from their glands and the regulation of hormone concentrations in the blood are mediated by internal control
mechanisms acting within the body. It should be understood that interactions
with external stimuli may also alter systemic hormone secretion. These mechanisms are especially important when dealing with environmental factors such
as stress, photoperiod and temperature variation. Exteroceptive mechanisms
that alter internal hormone secretion are mediated by the CNS. An external stimulus such as light from the environment can affect the endocrine system via photoreceptors in the eye, which, after transmission by the optic nerve to the brain,
can be transmitted via nerve tracts to the hypothalamus where the hypothalamic output of releasing or inhibiting factors and pituitary hormones is altered.
The release of pituitary hormones can alter thyroid or adrenal hormone release
(Fig. 3.7). Other external factors such as stress and temperature changes affect
the endocrine system in a similar manner, acting through external body receptors that relay messages to the brain and the hypothalamus, ultimately resulting in the increase or inhibition of hormone release.
46
Chapter 3
Fig. 3.6. Negative feedback loops of thyroid hormones on the hypothalamus and pituitary
gland. Thyroid hormones act in a ‘short-loop’ at the level of the pituitary or a ‘long-loop’
at the level of the hypothalamus.
Hormone Receptors
Effects of hormones on target tissues are mediated by receptors located in
the tissues affected by the respective hormones. Hormone receptors are cell
proteins, which bind to and modulate hormone effects in target cells/tissues.
They are highly specific for their hormone (ligand), are tissue-specific, present in very low concentrations in cells and have high affinity for hormones
(∼10−9−10−12 M). Receptors are the modulating molecules that confer tissuespecific actions on each hormone and mediate the cellular response to individual hormones. Receptors link the extracellular fluid environment of
blood and extracellular matrix (ECM) with the interior of the cell. Binding of
a hormone to its receptor sets in motion the sequence of stimulatory or
inhibitory events within the targeted cell, which is characteristic of that cell’s
function as well as the effect of the hormone which impinges upon it.
There are two fundamental receptor types, those present in the plasma
membrane of the cell and those which reside in the interior of the cell in the
cytoplasm or nucleus. Plasma membrane receptors bind protein, polypeptide and amino acid-derived hormones. The plasma membrane receptors act
as transducers, stimulating cytoplasmic second messengers which eventually affect gene expression in the nucleus of the target cell. Nuclear hormone
receptors bind ligands within the cell, in the nucleus or cytoplasm. Typical
ligands for intracellular receptors include small molecules that can easily
pass through the plasma membrane, such as steroids, thyroid hormones and
vitamin D. After binding their specific hormones, these receptors act directly
on binding sites in the nucleus to alter gene expression. Thus, these hormone
receptors are considered as ligand-activated transcription factors that regulate levels and rates of specific gene transcription.
Plasma membrane hormone receptors consist of three molecular
domains: an extracellular domain that binds the hormonal ligand, a
The Endocrine System
47
Fig. 3.7. Exteroceptive influence on hormone secretion.
lipophilic, transmembrane domain within the lipid bilayer and an intracellular cytoplasmic domain that activates intracellular signalling molecules.
These receptors can be classified into three types, based upon their structure,
associated kinases (enzymes that phosphorylate other proteins) and the second
messenger system activated by the receptor (Fig. 3.8). The intracellular domain
of the insulin and IGF-I receptors has an intrinsic tyrosine kinase (TK) activity,
which stimulates phosphorylation of the domain (autophosphorylation),
resulting in an active TK that, in turn, catalyses the phosphorylation of cytoplasmic substrates. This receptor is typical of insulin, IGF-I, epidermal growth
factor (EGF) and platelet-derived growth factor (PDGF). The second category of plasma membrane receptors is the cytokine receptors. Until they are
activated by hormone binding, cytokine receptors drift in the lipid bilayer as
48
Chapter 3
Fig. 3.8. Types of plasma membrane receptors.
single polypeptide monomers. Upon activation by hormone binding, these
receptors associate with one another and form dimers. Dimerization activates cytoplasmic Janus kinase (JAK), a kinase that phosphorylates proteins
on serine and threonine residues (a serine/threonine kinase). JAK binds to
the receptor and phosphorylates it and activates cytoplasmic second messengers called signal transducers and activators of transcription (STATs).
Cytokine receptors mediate the biological effects of GH, PRL, leptin and several interleukins. The last type of plasma membrane-associated receptors are
called the seven transmembrane-domain receptors, or G-protein-linked
receptors (GPLR). As the name implies, these receptors are snake-like proteins that wind in and out of the plasma membrane, with seven coils of the
molecule passing through the membrane. These are ubiquitous receptors,
which mediate the actions of many protein and amino acid-derived hormones, neurotransmitters and metabolites. Over 1000 of these receptors
have been identified to date. Among the hormones which act through these
receptors are TSH, parathyroid hormone (PTH), antidiuretic hormone
(ADH), LH/FSH, calcitonin, ACTH and catecholamines.
Effector Molecules, Transducers and Second Messengers
The intracellular response systems for plasma membrane-bound hormone
receptors function as ‘relay systems’ to convey a cell surface event (the binding of a hormone) to the interior of the cell, and eventually activate or silence
specific genes in the nucleus. This is accomplished by a series of phosphorylations (catalysed by kinases) and dephosphorylations (by phosphatases) of
cytoplasmic and nuclear proteins. All second messenger systems use phosphorylation/dephosphorylation events to increase or decrease enzyme
activity, thus altering the intracellular message activity. What follows is a
brief description of the cytoplasmic second messenger systems which mediate the effects of plasma membrane-bound receptors and their hormones.
The Endocrine System
49
The TK receptor family that binds insulin and IGF-I acts through two
distinct pathways (Fig. 3.9). The first pathway is believed to mediate mitotic
events. After tyrosine phosphorylation of the cytoplasmic protein, insulin
receptor substrate-1 (IRS-1) by the receptor, the primary effector, mitogenactivated protein (MAP) kinase, alters gene expression and activates mitogenic pathways. The pathway that activates glucose uptake by the cell
involves similar initial phosphorylations of cytoplasmic IRS-1 or IRS-2, but,
in this case, the phophatidylinositol-3 (PI-3) kinase pathway is activated.
This results in the movement of glucose transport molecules from
sequestered intracellular sites to the plasma membrane.
The insulin and IGF-I receptors are very similar in structure, but have
distinct hormone-binding specificities. The respective receptors have higher
affinities, or binding strengths, for their specific molecules. Both insulin and
IGF-I will bind to and activate the opposite receptor, but only at high concentrations. Thus, high levels of insulin can induce cell growth and mitosis
via the MAP kinase pathway and high concentrations of IGF-I can induce
the insulin-like effect of glucose transport via the PI-3 kinase pathway.
The GH/PRL receptor family consists of separate monomers which bind
to ligands and associate with another receptor monomer to form an active
receptor dimer (Fig. 3.10). Cytoplasmic JAK phosphorylates the receptor and,
via phosphorylation, activates STAT to induce nuclear gene transcription.
Alternatively, the activated receptor and JAK2 can stimulate the
shc/Ras/MAP kinase pathway to induce gene expression and mitosis. Lastly,
a minor pathway mediates transient insulin-like effects of GH on glucose
transport into target cells via stimulation of the IRS/PI-3 kinase pathway.
The seven transmembrane-domain receptors, or GPLR, act through
plasma membrane-bound G-proteins inside the cell which, in turn, activate
other intracellular messengers. After ligand binding to the receptor, the
receptor associates with and activates G-proteins. The G-proteins are bound
Fig. 3.9. Intracellular signalling pathways of the insulin and IGF-I receptor system.
50
Chapter 3
to the cytoplasmic surface of the plasma membrane. They are guanidine
triphosphatases (GTPases) that hydrolyse GTP to GDP, releasing phosphate.
The G-proteins are heterotrimeric molecules, consisting of three subunits: α, β and γ. Functionally, there are only two subunits, the α subunit and
(βγ) subunit, as the latter are tightly bound to one another. The α subunit of
the G-proteins contains the nucleotide-binding site, which is inactive when
GDP is bound to it. Contact with a hormone-activated receptor causes the
release of GDP, which is immediately replaced with GTP. The GTP is rapidly
hydrolysed to GDP. The active, GTP-bound form of the G-protein acts as a
molecular switch that activates effector molecules such as adenylate cyclase
(AC) and phospholipase-C (PLC) within the cell. As the GTP-bound form of
the G-proteins is present only for a brief time, the activation of effector molecules is rapid and transient.
The GPLR act through different effector molecules, depending
upon the receptor, its ligand, the cell type and the G-linked protein. The Gproteins activate (or inhibit) AC or PLC. AC is bound to the cytoplasmic face
of the plasma membrane and exists as ten isoforms. Activation of AC by
G-proteins stimulates the formation of cyclic AMP (cAMP) from ATP. All
receptors that act via cAMP use the stimulatory G-protein (Gsα) of the α subunit to activate AC. Another α subunit isoform, the inhibitory G-protein
(Giα), inhibits AC. Elevated intracellular concentrations of cAMP bind to
and activate the enzyme protein kinase-A (PKA) (Fig. 3.11). PKA is a serine/threonine kinase and alters the activities of other intracellular proteins
Fig. 3.10. Intracellular signalling pathways of the GH/PRL receptor.
The Endocrine System
51
by adding phosphates (from ATP) to their serine or threonine residues. The
protein substrates for PKA are variable, and differ with cell type. In skeletal
muscle, upon stimulation by epinephrine, PKA phosphorylates and activates enzymes involved in glycogen breakdown, while inactivating
enzymes involved in glycogen synthesis. This results in a rapid increase in
glucose availability. The phosphorylation of the cAMP regulatory-binding
protein results in its activation, diffusion to the nucleus and binding to
cAMP regulatory elements (CRE) of specific genes. In this way, hormonal
stimulation of a cell alters the transcription of specific genes.
Activation of the plasma membrane-bound enzyme, PLC by GPLR
provides an alternate pathway for the intracellular effects of hormones in specific tissues. Another isoform of the G-protein α subunit, the Gqα subunit, activates PLC. PLC exists as three forms: β, γ and δ and there are four isoforms of
the β form (β1–β4). PLC-β mediates many of the effects of GPLR. PLC-β acts
on a plasma membrane-bound inositol phospholipid, phosphatidylinositol
(4,5)-bisphosphate (PIP2), to form inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG) (Fig. 3.12). The products of this reaction, IP3 and DAG, mediate
separate intracellular processes. IP3 diffuses through the cytoplasm to the SER.
Here, it binds to an IP3 receptor/calcium channel and stimulates Ca2+ influx
into the cytoplasm from storage sites in the endoplasmic reticulum (Fig. 3.13).
The elevated cytoplasmic concentrations of calcium bind to protein kinase-C
(PKC) and stimulate its migration to the plasma membrane, where DAG
remains bound. The most important role of DAG is to activate PKC, which in
turn, phosphorylates additional cell-specific cytoplasmic proteins, resulting in
their activation or repression.
Fig. 3.11. Activation of the cyclic AMP pathway by G-protein-linked receptors.
52
Chapter 3
Fig. 3.12. Conversion of plasma membrane phosphatidylinositol (4,5)-bisphosphate
to the intracellular messengers diacylglycerol and inositol 1,4,5-triphosphate.
The steroid hormone receptor family belongs to the superfamily of
steroid/nuclear receptors, which are located in either the nucleus (thyroid hormones) or cytoplasm (glucocorticoids and sex steroids). These are ligand-activated nuclear transcription factors, which bind directly to promoter regions of
specific genes in the nucleus. The steroid receptors consist of four functional
domains which are responsible for hormone binding, nuclear localization,
DNA binding and activation of transcription (Fig. 3.14). The glucocorticoid and
oestrogen receptor molecules are transcription factors whose DNA-binding
domain consists of ‘zinc-fingers’ (leucine-rich areas that contain zinc) that bind
DNA and mediate the activity of these steroids on gene transcription.
There are four classes of the steroid/nuclear receptor superfamily. The
first class of steroid receptors associate with one another to form homodimers,
which bind to two palindromically arranged, hexanucleotide half sites on the
gene promoter. The receptors for glucocorticoids, progesterone, oestrogen and
androgens are members of this class. The second type of receptor, retinoid ×
receptors (R × R), forms heterodimers with unrelated steroid receptors and
bind to direct repeat sites on the DNA promoter. The receptors for thyroid hormones and 1,25-dihydroxy vitamin D3 belong to this class of steroid receptor.
Dimorphic orphan receptors are so-called because their ligands are mostly
The Endocrine System
53
Fig. 3.13. Activation of the phosphoinositol pathway by G-linked protein receptors.
unknown. The last class of steroid receptors is called monomorphic orphan
receptors, which have been noted, but whose ligands are unknown.
An overview of the actions of the endocrine system and hormones is
given in Fig. 3.15, which outlines the pathways involved in the actions
of protein and steroid hormones. This includes secretion from the
endocrine glands into the bloodstream, transport via the systemic circulation,
interaction with specific receptors in target cells and activation of intracellular
A/B
Regulatory
domain
C
D
E
DNAbinding
Nuclear
domain
targeting
domain
Hormone-binding
domain
(A) Steroid receptor gene
Domain E
hormone binding
Domain A/B
nuclear interaction
Domain D
(B) Steroid receptor protein
Fig. 3.14. Steroid receptor gene and protein domains.
Domain C
DNA-binding
54
Chapter 3
Fig. 3.15. Summary of hormone action – from gland to target DNA.
messenger systems and activation or repression of specific nuclear genes.
The culmination of hormonal stimulation of a target cell results in the alteration of specific physiological processes in the target tissue.
References and Further Reading
Gomperts, B.D., Kramer, I.M. and Tatham,
P.E.R. (2002) Signal Transduction.
Academic Press, New York, 424 pp.
Hadley, M.E. (1984) Endocrinology. PrenticeHall, Englewood Cliffs, New Jersey,
547 pp.
Pineda, M.H. and Dooley, M.P. (eds) (2003)
McDonald’s Veterinary Endocrinology and
Reproduction, 5th edn. Iowa State Press,
Blackwell, Ames, Iowa, 597 pp.
Squires, E.J. (2003) Applied Animal Endocrinology. CAB International, Wallingford, UK,
272 pp.
Starling, E.H. (1905) The chemical correlation
of the functions of the body. Lancet ii,
339–341.
Turner, C.D. and Bagnara, J.T. (1976)
General Endocrinology, 6th edn. W.B.
Saunders, Philadelphia, Pennsylvania,
596 pp.
4
Development of Muscle, Skeletal
System and Adipose Tissue
The focus of this book is on the hormonal regulation of the growth and
development of the three tissues that are essential to the whole body development and composition of animals: muscle, bone and fat. The purpose of
this chapter is to examine the normal growth and development of these three
tissues. The effects of external agents, such as hormones, on the growth and
metabolism of these tissues will be covered in subsequent chapters.
Embryonic Development and Cell Differentiation
As we have seen, development encompasses the alteration of body form. It
also involves differentiation of cells into cells that assume specialized forms
and functions, resulting in distinct tissues and organs which are capable of
the processes we associate with animals. These differentiated cells have
functions that range from gas exchange to reproduction, locomotion and
consciousness.
The development of all animals begins with a single, undifferentiated
cell, the diploid zygote (Fig. 4.1). The zygote results from the fertilization of
a haploid oocyte by a haploid spermatozoon. This single cell will undergo
mitosis within the zona pellucida, a gelatinous acellular layer surrounding
the early oocyte and embryo. Cell replication results in a doubling of cell
numbers with each round of mitosis. The replication of these undifferentiated cells forms of a ball of eight to 64 cells called a morula. The earliest differentiation of the embryo occurs when there are 128 to 256 cells, depending
upon the animal species. At this time, a fluid-filled cavity, the blastocoele,
forms and the embryo is called a blastocyst. The blastocyst is distinguished
by two distinct groups of cells, the inner cell mass and the trophoblast. The
inner cell mass contains cells destined to form the embryo proper and this
cell aggregate is segregated to one side of the blastocoele. The trophoblast
cells, surrounding the blastocoele, comprise the remainder of the blastocyst.
The trophoblast will form the placenta and the embryonic membranes such
as the amnion, chorion and allantois. After escape from the zona pellucida,
the blastocyst attaches to the epithelium of the uterus and makes a physical
connection to the maternal circulation via the placenta.
©K.L. Hossner 2005. Hormonal Regulation of Farm Animal Growth (K.L. Hossner)
55
56
Chapter 4
Fig. 4.1. Development of the early embryo after fertilization.
Traditionally, only the cells of the very early embryo, i.e. the first eight or
16 cells of the embryo, have been considered totipotent. In other words, only the
undifferentiated, early embryonic cell was capable of forming a complete
organism. In general, after cells had undergone differentiation, they were
irreversibly altered and incapable of dedifferentiation into a more universal
cell type capable of forming a functional organism. It is now known that
most cells of the body, even in mature animals, under the proper conditions
in vitro, can be induced to undergo dedifferentiation and are subsequently
capable of forming entire living organisms through the process of cloning.
Pluripotent cells or cell types, such as the mesoderm, are capable of limited
formation of different tissues, and can differentiate into more than one cell
type, such as muscle, bone or fat. They cannot form an entire organism.
After attachment to the uterus, embryonic cells multiply, migrate and
differentiate to form three germ layers (Fig. 4.2), by a process called gas-
Muscle, Skeletal System and Adipose Tissue
57
Fig. 4.2. Cross-section through an early developing embryo showing the three germ layers.
trulation. During embryonic development, the three germ layers differentiate into the specific organs and tissues of the animal. The outermost of these
layers is called the ectoderm and it will form the brain, spinal cord and skin
of the animal. The inner layer is called the endoderm and it will form many
of the internal organs of the animal, including most of the gastrointestinal
system. The middle layer of cells in the embryos is called the mesoderm, and
it is this layer which gives rise to three tissues that are the primary focus of
this book, bone, muscle and fat, as well as cartilage, blood, dermis and
connective tissues such as tendons, ligaments and joint capsules.
The paraxial mesoderm, lying on either side of the embryonic neural
tube, is a specialized type of mesodermal tissue which forms the embryonic
somites (Figs. 4.3 and 4.4). The somites consist of segmented blocks of cell
condensations which form sequentially along both sides of the embryonic
neural tube (future spinal cord) and notochord (an embryonic support structure beneath the neural tube) during embryogenesis. The somites form
sequentially in an anterior to posterior (head-to-tail) sequence, with the
somites closest to the head forming first. Different areas of the somites will
differentiate into different structures of the body. The ventral portion of the
somite is called the sclerotome. It will differentiate into the bones of ribs and
vertebrae. The rest of the somite, called the dermomyotome, is divided into
three developmental regions. The dorsal dermomyotome will form some
skeletal muscles and the dermal skin of the trunk. The epaxial dermomyotome will form the epaxial musculature of the back. The ventrolateral portion of the somite will give rise to the hypaxial musculature of the trunk and
limbs. When muscle formation is initiated, the progenitor muscle cells of the
somites (myoblasts) detach from the body of the somite and migrate to
specific areas of muscle formation, such as the embryonic limb buds.
Another type of mesoderm is called the mesenchyme, a type of loosely
packed aggregation of mesodermal cells (mesenchymal cells), suspended in
the gelatinous ECM located between the ectodermal and endodermal germ
layers. These cells are motile, in some cases contain migrating cells from
the somites. Mesenchymal cells migrate through the body to form many of
the skeletal, connective tissue, blood and smooth muscle systems. Another
type of important cell derived from mesoderm is the fibroblast. These cells are
widely distributed in the embryo and the adult. Fibroblasts form connective
58
Chapter 4
Fig. 4.3. Diagram of a 5 mm pig embryo showing relationships between somites, anterior limb
bud and spinal cord.
Fig. 4.4. Cross-section through an embryo showing the somites at the levels of the limb bud.
Muscle, Skeletal System and Adipose Tissue
59
tissue cells that produce and secrete the ECM. They are involved in the
growth, wound repair and accessory physiological processes of all tissues.
They are relatively undifferentiated cells and, under appropriate conditions,
are capable of differentiating into osteoblasts and chondroblasts in the adult.
Cell Proliferation and Differentiation
Differentiation of cells involves the assumption of a specialized function. For
example, differentiated cells may be capable of contraction (of muscles),
lipid storage (adipose) or the propagation of a nerve impulse, the formation
of an idea. These functions are accompanied by the expression of specific
genes for specific products related to cell function such as the genes for contractile proteins in muscle or lipogenic genes in adipose tissue. Cellular differentiation follows a generalized pattern (Fig. 4.5), which is applicable to all
tissues. An undifferentiated cell undergoes a round of mitotic proliferation,
which results in two daughter cells. One of these cells, in turn, is committed
to undergo differentiation in which the cell assumes an essentially irreversible specialized function. Accordingly, in many cases, the second daughter cell remains undifferentiated, and becomes mitotically quiescent. This
reserve stem cell provides a reservoir of cells, which can be activated under
appropriate conditions when additional differentiated cells are required. The
sequence of mitotic proliferation followed by differentiation is generally,
although not always, a mutually exclusive event. Proliferating cells are
devoting their metabolic energy and genetic resources towards increasing
cell numbers. It is only upon appropriate stimuli that these cells alter their
functions, cease cell division and begin the processes associated with differentiation.
Fig. 4.5. General pattern of cell differentiation.
60
Chapter 4
The Skeletal System and Bone Growth
The importance of bone tissue to animal agriculture is illustrated by the
costly problems associated with bone dysfunction. Recent changes in the
genetics and nutrition of livestock animals have led to increased growth
rates, mature animal sizes and weights. Associated with these fundamental
alterations of animal metabolism and physiology is an increased incidence
of long bone anomalies. Modern domesticated animals are beset with an
array of skeletal anomalies associated with their intense selection for rapid
growth. For example, half of the broiler chickens in the USA are affected by
tibial dyschondroplasia, in which growth plate cartilage accumulates in the
tibia and femur, leading to lameness and increased leg fractures. A similar
problem in swine, osteochondrosis, is characterized by a lack of bone formation (ossification) in the growth plate of the load-bearing bones. These
problems lead to losses of millions of dollars to the livestock industry and it
is important to understand the growth and development of the important,
yet often overlooked, skeletal system. Traditionally, a great deal of attention
has been paid to the obvious components of body composition in farm animals, the skeletal muscle and fat. The increased body size, altered composition and rapid growth rates of modern farm animals require a reordering of
our priorities. A basic knowledge of bone growth and development, and
its regulation, are essential for the optimal management and successful
production of food animals.
Bone is a dynamic, living tissue which has several functions that are
important to the metabolism, growth and normal functions of animals. Most
obviously, bone provides a structural support for the body, providing attachment points for muscles to allow for body movements. The high strength to
weight ratio of bone is essential to provide mobility for the animal. Of
course, bone must also be flexible, providing some elasticity to the movements of animals. Bone also has important metabolic functions that are
important for whole body homeostasis. It provides a reservoir for several
minerals including calcium, phosphate, magnesium, sodium and carbonate
in the body. These minerals are in equilibrium with blood minerals and are
mobilized from or deposited in the bone matrix depending upon the physiological requirements of that animal. Another essential function of bone is the
production of blood cells by bone marrow. Marrow is also the site of production of stem cell precursors that will form osteoblasts and osteoclasts of
bone. Recent observations suggest that bone marrow may also be the source
of stem cells for other tissues, such as skeletal muscle and adipose tissue.
Thus, bone plays important roles in several vital processes associated with
the animal’s well-being. Bone has not only a purely mechanical and structural role, but also plays essential physiological roles in the maintenance of
electrolyte homeostasis and the production of stem cells. Like cartilage, bone
is a highly specialized type of connective tissue. The ECM of bone consists
primarily of mineralized crystalline deposits of calcium and phosphate, and
forms the majority of the bone mass. This extensive ECM surrounds the relatively sparse bone cells embedded in this rigid matrix.
Muscle, Skeletal System and Adipose Tissue
61
Types of Bones and Gross Anatomy
There are two types of bone, based on their embryonic origins. Membranous
bone (also called intramembranous bone) is formed de novo from bone cells.
Examples of membranous bone are the flat bones of the skull, i.e. the calvarium, or skullcap of the head. Calvarial osteoblasts are derived directly from
precursor mesenchymal stem cells. The second type of bone is cancellous
bone. Most bones, including those of the appendages, are cancellous bones.
Also called endochondral bone, this type of bone is formed by replacing an
existing cartilage model with bone. This model is formed when embryonic
mesenchymal cells proliferate, undergo hypertrophy and form condensations of cells at the site of future bone formation. The mesenchymal cells differentiate into chondroblasts that deposit the ECM of cartilage, forming a
miniature cartilaginous model of the future bone. When completely surrounded by matrix, they are then called chondrocytes. Chondrocytes proliferate, differentiate and hypertrophy and secrete a cartilage matrix that is
mineralized.
The long cancellous bones of the skeleton have a characteristic gross
anatomy which relates to the function of these bones (Fig. 4.6). This includes
the central shaft, which is called the diaphysis (pl. diaphyses). Long bone ossification in the embryo begins in the central region of the diaphysis where
ossification of cartilage first occurs. The inner layer of the long bone, which
surrounds the marrow, is called the endosteum. Osteoblasts and osteoclasts
responsible for growth and remodelling of the bone are found here. The
outer layer of the long bone, the periosteum, contains osteoblasts, osteocytes
and fibroblasts. This is where the increase in bone diameter occurs. The end
of the bones is called the epiphysis (pl. epiphyses). This is the secondary centre of ossification, where long bone mineralization continues in the embryo.
The epiphyseal plate, or growth plate, is the strip of cartilage between the epiphysis and the diaphysis. The epiphyseal plate contains chondroblasts and
chondrocytes, which form the cartilage template for linear bone growth. The
portion of the long bone between the diaphysis and the epiphysis is called
the metaphysis. This is where bone mineralization of the cartilage matrix
occurs. The epiphyseal plate is generally fused (ossified) around the time of
puberty when linear growth ceases.
Bone Cells and Bone Matrix Formation
Bone consists of three major types of cells with different functions: osteoblasts,
osteoclasts and osteocytes. Osteoblasts are derived from mesenchymal cells
during embryonic development and from mesenchymal stem cells in the
periosteum or stromal stem cells in the marrow (endosteum) after bone formation. The precursor cells become committed to the osteoblast cell lineage,
proliferate and differentiate into preosteoblasts. Preosteoblasts are committed
to differentiate into osteoblasts. Osteoblasts are precursors of mature osteocytes. Osteoblasts are fully differentiated cells that are responsible for the
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Chapter 4
Fig. 4.6. Anatomy of a long bone.
production of bone matrix. Osteoblasts display a cell phenotype which is
typical of a protein-producing cell. They contain extensive RER and a welldeveloped Golgi apparatus. The primary function of the osteoblasts is to
secrete the organic ECM, or osteoid. Initially, the osteoid is not mineralized
but serves as a template for mineralization. In addition to secretion of collagen, the osteoblasts are also the source of extracellular collagenase.
Collagenase is secreted during bone remodelling to degrade the collagen
portion of the organic matrix. Collagenases exist in three isoforms (collagenase 1, 2 and 3), all of which degrade Type I collagen in the matrix with similar efficiencies. Secretion of collagenases is stimulated or inhibited during
different developmental stages and during bone remodelling and repair.
The organic matrix of bone consists primarily of Type I collagen
(90–95%) with the remainder composed of other proteins and proteoglycans.
Proteoglycans are large molecular complexes consisting of 95% carbohydrates and 5% proteins. Along with collagen, many minor proteins and glycoproteins are secreted by osteoblasts into the osteoid. These include
proteoglycans such as decorin and biglycan. Both bind to chondroitin or dermatin sulphate. Decorin binds to (decorates) collagen fibrils while biglycan
occupies the pericellular area of osteoblasts and osteocytes. Transforming
growth factor-β (TGF-β) binds to both of these molecules. Decorin and biglycan are believed to act as matrix organizers, helping to orient the collagen
molecules. Other matrix proteins contain the arginine, glycine, aspartic acid
(RGD) sequences that bind to cell surface integrin receptors to provide
matrix–cell interactions and adhesion. These include fibronectin and throm-
Muscle, Skeletal System and Adipose Tissue
63
bospondin. The PDGF growth factors also bind to thrombospondin and
osteonectin. Another matrix protein, osteonectin, binds calcium, hydroxyapatite and several collagen isoforms (I, II and IV). Osteonectin is thought to
act as an anti-adhesive component, reducing cell adhesion to the matrix and
altering cell morphology. Osteopontin is a glycoprotein that contains the
RGD sequence for integrin binding. It is secreted by osteoblasts that are
nearing maturity. Its deposition leads to the first mineralization of the matrix
and thus, serves as a marker for osteoblast maturation and bone mineralization.
Osteoblasts also secrete hyaluronic acid, a large carbohydrate that forms
a portion of the ECM. It consists of a repeating disaccharide of glucuronic
acid and N-acetylglucosamine. It is often found in association with multiple
core proteins that bind to the long axis of hyaluronic acid, forming a bottlebrush structure. As with many carbohydrates found in proteoglycans,
hyaluronic acid contains acetylated amino sugars. For this reason these
carbohydrates are called glycosaminoglycans. Hyaluronic acid and other
glycosaminoglycans are able to bind water up to 10,000 times their own volume to yield gels and thus, to capture space for further matrix deposition.
Other glycosaminoglycans include the chondroitin sulphates, polymers of
glucuronic and N-acetyl sugars and keratin sulphate, a sulphur-rich minor
protein component.
During the development of the osteoblast, there is a distinct sequence of
events that characterize its differentiation. In the early stages, osteoblasts
undergo cell mitosis and proliferation. The first components of osteoid
secreted by the osteoblasts are Type I collagen and fibronectin. As the cell
continues to mature, they continue to secrete collagen and begin to produce
alkaline phosphatase. Alkaline phosphatase is an enzyme complex that is
often used as a molecular marker for osteoblast differentiation. Alkaline
phosphatase hydrolyses pyrophosphate and ATP, both inhibitors of matrix
calcification. This allows the initial formation of hydroxyapatite crystals on
the osteoid. Later in the life cycle of the osteoblast, collagen production
slows and osteocalcin and osteopontin are secreted.
The mineralization of the organic matrix to form the rigid structure we
know as bone is regulated by osteoblasts, although the mechanisms are not
completely understood. High local concentrations of Ca2+ and PO 43– are
required for the formation of calcium phosphate precipitates. Mature
osteoblasts secrete membrane-bound matrix vesicles that bud from cytoplasmic processes of these cells. These vesicles are rich in alkaline phosphatase and allow the precipitation of calcium phosphate and the
crystallization of hydroxyapatite molecules [Ca10(PO4)6(OH)2] on to the
organic matrix. Collagen serves as a template for mineral deposition and
may regulate the process of mineralization. The inorganic, insoluble hydroxyapatite forms the mature bone shape and function.
After osteoblasts are completely surrounded by mineralized matrix,
they differentiate into osteocytes. The osteocytes are thought to be responsible for the maintenance of the bone. Osteocytes continue to produce matrix
proteins and are capable of limited bone resorption. Osteocytes reside in
pockets in the bone called osteocytic lacunae, surrounded by the mineralized
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Chapter 4
ECM of the bone (Fig. 4.7). Deposition of mineral makes the matrix impermeable and specialized channels in the bony matrix, called canaliculi, allow
osteocytes to physically connect each other. Osteocytes fill the canaliculi
with long cytoplasmic projections, which form gap junctions with adjacent
osteocytes. These specialized connections allow intercellular communication
between the osteocytes. The canaliculi and the lacunae contain an extracellular fluid space between the plasma membrane and the bone matrix. This
fluid, which moves throughout the network of osteocytes, is the only source
of nutrients for the osteocyte.
Osteoclasts and bone resorption
Osteoclasts are specialized bone cells responsible for the resorption of mineralized bone. These cells are large, multinucleated phagocytes, containing
four to 29 nuclei. Osteoclasts are derived from the fusion of circulating
mononucleated blood cells (monocytes) derived from the bone marrow.
They reside on the surfaces of the bone, primarily in the marrow and periosteum, and play an essential role in bone resorption that is required for
growth, remodelling and fracture repair of bones. Osteoclasts are characterized by an abundance of mitochondria, Golgi apparati and lysosomes.
Fig. 4.7. Relationship of the primary bone cells.
Muscle, Skeletal System and Adipose Tissue
65
The most conspicuous characteristic of the osteoclasts relates to their boneresorbing function (Fig. 4.8). The plasma membrane of osteoclasts contains
multiple, deep folds on the cell side that faces the bone surface called a ruffled border. Osteoclasts are tethered to the bone surface by an encircling
zone of attachment called the sealing zone. The sealing zone acts to attach
the osteoclast to the bone matrix and to isolate the underside of the osteoclast from the external environment. The sealing zone attachment occurs via
integrin receptors on the osteoclast plasma membrane that bind to RGD
sequences in matrix proteins. This forms a bone-resorbing compartment
beneath the osteoclast where bone matrix can be degraded. Bone resorption
by osteoclasts occurs by acidification and solubilization of hydroxyapatite
crystals and by proteolysis of matrix proteins within the sealing zone. The
osteoclast produces H+ via the action of carbonic anhydrase within the cell.
The protons are pumped across the ruffled border membrane by proton
pumps, reducing the pH of the sealing zone. The osteoclast also secretes a
variety of lysosomal and non-lysosomal proteases into the bone-resorbing
compartment to degrade the matrix. Lysosomes provide the protease,
cathepsin K and acid phosphatase. Non-lysosomal proteases include the
matrix metalloproteinases, collagenase and gelatinase B. This combination
of enzymes and protons acts to dissolve the bone matrix and results in small
‘craters’ on the surface of the bone, which can be remodelled or replaced
with new matrix.
Regulation of osteoclast function
The activities of osteoclasts are regulated by osteoblasts. Receptors for most
external factors, such as hormones and growth factors that induce bone
Fig. 4.8. Components of osteoclast attachment and bone dissolution.
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Chapter 4
resorption, are found in osteoblasts, not osteoclasts. Osteoblasts also produce
growth factors and cytokines that influence the differentiation and function
of osteoclasts, either by direct paracrine actions or after deposition into the
matrix. The effects of osteoblasts on osteoclast differentiation and function
are mediated by a recently discovered receptor–ligand system, called the
RANK/RANKL system (Fig. 4.9). This system is composed of three components. The membrane-bound receptor activator of NFκB (RANK) receptor, a
member of the tumour necrosis factor (TNF) receptor family, is present in
osteoclast progenitor cell plasma membrane. The plasma membrane of
osteoblasts contains the immobilized RANK transmembrane ligand
(RANKL) that binds to and activates the RANK receptor molecule when
osteoblasts and osteoclast precursor cells are in contact with one another. In
addition, osteoblasts secrete a soluble decoy receptor called osteoprotegerin
(OPG) that binds to and inactivates RANKL. NFκB is the cytoplasmic mediator of RANK receptor in osteoclasts. Cell-to-cell contact between osteoblast
and presumptive or mature osteoclasts allows binding and activation of the
Fig. 4.9. The RANK, RANKL, OPG system of osteoblasts and osteoclasts.
Muscle, Skeletal System and Adipose Tissue
67
RANK/RANKL system. This induces the differentiation and activation of
osteoclasts and bone resorption. The negative regulation of osteoclast formation occurs by osteoblastic secretion of OPG. OPG acts as a decoy receptor, binding RANKL and inhibiting its binding to RANK, reducing the
differentiation of osteoclasts. As a result of RANK/RANKL interaction,
formation of osteoclasts and thus, bone resorption is enhanced.
The Epiphyseal Plate
The anatomy of the epiphyseal plate and the relationships of the cells
involved in long bone growth are shown in Fig. 4.10. Chondrocytes are
arranged in columns that are maintained by mineralization of the spaces
between the columns. Mineralization of the cartilaginous matrix begins in
the zone of proliferating chondrocytes, farthest from the growth plate, and
becomes denser around the hypertrophic chondrocytes. Some mineralized
cartilage is resorbed at the marrow cavity margins by osteoclasts to provide
room for new bone deposition and vascular growth. The remaining mineralized cartilage provides the surface for osteoblasts to differentiate and
mineralize the ECM of bone.
Fig. 4.10. Anatomy of the epiphyseal plate and metaphysis.
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Chapter 4
Skeletal Muscle Development
Along with the skeletal system, skeletal muscle provides the animal with the
physical support to remain upright and the locomotory skills to move, run
and feed. The diaphragm is also composed of skeletal muscle, and thus,
skeletal muscle is essential for the regulation of respiration in the animal.
Muscle is, of course, the primary product of meat animal production, forming the basis for an extensive animal agriculture production system and one
of the primary sources of dietary protein in many cultures. In many animals,
the majority of skeletal muscle development occurs during embryonic development. This provides the newborn animal with immediate mobility, allowing it to accompany adults and to avoid predation. Many farm animals ‘hit
the ground running’ and are capable of standing, nursing and walking
immediately after birth.
Skeletal muscle is a tissue that is specialized to provide contraction
forces for movement. Mature skeletal muscle is unique in that it is composed
of multinucleated, non-mitotic cells containing large quantities of large,
insoluble proteins that provide muscle strength, elasticity and contraction.
Muscles function in conjunction with the nervous system, which provides
the stimulus for muscle contraction. Muscle is also dependent upon its interaction with the skeletal system, which provides the framework for muscle
attachment and the support for muscle movement and, subsequently,
together the skeleton and the skeletal muscle aid in the maintenance of body
form and shape. Compared to the skeletal system, the nomenclature and
interaction of the cells involved in skeletal muscle formation are relatively
straightforward.
Embryonic formation of skeletal muscle
Skeletal muscle of the body and limbs is derived from mesodermal cells
which differentiate into the muscle progenitor cells, the myoblasts.
Myoblasts are mononucleated cells, which are capable of mitosis and fusion
with other myoblasts to form multinucleated cells. Myoblasts accumulate
small amounts of the myofibrillar proteins actin and myosin during maturation, but myoblasts are not contractile cells. Myoblasts fuse with one another
to form the embryonic myotubes, characterized by the presence of multiple,
centrally located nuclei within each cell. A single myotube may contain hundreds of nuclei. After fusion of myoblasts to form myotubes, the synthesis of
myofibrillar proteins is accelerated. The mature differentiated muscle cell is
called a muscle fibre or a myofibre. Myofibres are characterized by additional actin and myosin accumulation, an increase in cell size and the migration of cell nuclei to a peripheral location, making room for the accumulating
contractile apparatus of the myofibre.
In vertebrate animals, mesodermal somites of the embryo give rise to the
muscles of the limbs and body, while local condensations of mesodermal
cells form the skeletal muscle of the head. As described earlier, the somites
Muscle, Skeletal System and Adipose Tissue
69
are paired condensations of mesoderm that form along both sides of the
embryonic spinal column. The hypaxial musculature of the limbs and body
wall is derived from the myogenic precursor cells residing in the lateral portion of the somites. The epaxial musculature of the back and intercostal (rib)
muscles is derived from the medial halves of the somites. Embryonic limb
muscle myogenesis begins shortly after the formation of the three primary
germ layers (a process termed gastrulation), in the first third of gestation,
when myogenic progenitor cells in the somites adjacent to the limb buds
cells delaminate, or detach from the somites. This is followed by the migration of these cells to the sites of muscle formation, for example, the embryonic limb buds. In the limb buds, myoblasts form dorsal and ventral
aggregates of cells surrounding the cartilage primordia that will later form
endochondral bone. Genes that regulate muscle formation are activated and
the myoblasts begin to actively proliferate. This ensures that there is an adequate population of myoblast progenitors capable of fusing to form mature,
functional skeletal muscle. Myoblast proliferation is followed by cessation of
mitosis and differentiation into myotubes. In this process, myoblasts fuse
with one another to form multinucleated myotubes that synthesize large
amounts of the contractile proteins (Fig. 4.11). Embryonic myoblasts fuse
with one another to form primary myotubes, which defines the type, shape
and location of muscles in the adult. During the second third of gestation,
fetal myoblasts form secondary myotubes, which form around the primary
myotubes. The secondary myotubes undergo hypertrophy and attach to
other myotubes via tight connections called gap junctions, allowing cellto-cell communication between the myotubes. The fetal myoblast period,
when secondary muscle fibres are formed, determines the ultimate number
of fibres in the adult. Myofibre formation is generally completed during the
first two-thirds of prenatal development, although this process is speciesdependent.
Skeletal muscle satellite cells
Skeletal muscle is capable of limited repair and regeneration and grows
throughout adulthood. Both muscle DNA content and muscle mass increase
during the postnatal period. It is estimated that 70% to more than 90% of
myofibre DNA accumulates postnatally. This occurs despite the observations that myofibre numbers are fixed at birth and myofibres are incapable
of mitosis. As mature myofibres are incapable of mitosis, the mechanism of
postnatal myofibre DNA accumulation was unknown until relatively
recently. In the 1960s, muscle satellite cells were discovered. Satellite cells are
considered the adult equivalent of embryonic myoblasts and provide new
nuclei to growing or damaged skeletal muscle fibres. These cells lie just outside the cell membrane of the mature myofibre, between the cell and underlying the basement membrane. These tiny, mononucleated cells consist
almost entirely of nuclei, with very little cytoplasm. Only a few satellite cells
are present in muscle. It has been estimated that satellite cells account for 1%
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Chapter 4
Fig. 4.11. Differentiation of muscle fibres.
to 10% of the nuclei present in muscle fibres. Upon activation, satellite cells
proliferate, fuse with each other and with the myofibre sarcolemma and add
more nuclei to the myofibre. Some of the satellite cells resulting from replication return to quiescence and retain their extracellular site to assure that
the pool of progenitor cells is not depleted. Fusion of satellite cells with
mature muscle fibres results in an increase in adult muscle DNA content that
can support an increased muscle mass. In the large, multinucleated muscle
fibre, it is thought that a nucleus has only a limited sphere of influence and
can affect only the cytoplasm immediately around it. As the myofibres are
enormous cells, spanning up to 100 µm in length, multiple nuclei are
required to provide the synthetic machinery needed to increase muscle protein synthesis that must accompany cell growth. Fusion of satellite cells with
adult muscle fibres provides a mechanism to increase the DNA content of
the non-mitotic muscle cell, without nuclear replication.
Muscle, Skeletal System and Adipose Tissue
71
Satellite cells and myoblasts share many things in common. Both are
myogenic cell precursors that can undergo mitosis and fusion to form the
multinucleated myofibres and it has been suggested that satellite cells are
simply adult myoblasts. There are several lines of evidence that myoblasts
and satellite cells are distinct from one another and are not simply different
developmental stages of the same cells. The most obvious difference
between these cell types is, of course, developmental. Satellite cells are postnatal stem cells, while myoblasts are present during embryonic and fetal
development. In contrast, it has been shown that myotubes derived from
satellite cells contain ∼1/3 of the actin per myotube nucleus than myoblastderived myotubes. Tumour-promoting phorbol esters have different effects
on these cells. They block myoblast differentiation, but do not affect the differentiation of satellite cells. Acetylcholine receptor channels are present in
satellite cells but are seen only after differentiation in myoblasts. In addition,
myoblasts are active in DNA synthesis and mitosis, while satellite cells are
primarily quiescent cells that undergo mitosis only upon activation. Finally,
satellite cells are much smaller than myoblasts. Thus, satellite cells appear to
be a separate population of adult stem cells, which are morphologically and
functionally distinct from fetal and embryonic myoblasts. Satellite cells are
derived from adult muscles and used in primary cultures to study muscle
growth and differentiation. A number of immortalized myoblast cell lines
are used as experimental models to study myogenesis. Examples of these
include the L6, C2C12 and C2 myoblast cell lines.
Myofilaments, Myofibrils and Sarcomeres
Mature muscle cells are composed of contractile proteins arranged into functional contractile units called myofibrils. Myofibrillar proteins account for
the majority of proteins in the muscle cell, comprising 55% to 65% of the total
protein in a myofibre. Myofibrils are long threads of protein composed of
multiple repeating sarcomeres, connected end-to-end. Myofibrils are massive bundles of myofilaments, about 1 to 2 µm in diameter and extending the
length of the muscle fibre, up to several millimetres. There are thousands of
myofibrils in each muscle cell, arranged side-by-side and connected to each
other with proteins called intermediate filaments. The intermediate filaments provide physical support for the myofibrils and join the myofibrils
into a single cohesive unit that provides a unified contraction force. A major
intermediate filament, often used as a marker for skeletal muscle, is the protein desmin. Sarcomeres are the functional contractile unit of the muscle and
are made up of the two major myofilament proteins, actin and myosin. Actin
and myosin generate the contraction forces within the sarcomere. Alteration
of the thick myosin filaments with the thin actin filaments within the sarcomere give the skeletal muscle its typical striated, or striped, appearance.
The anatomy of a sarcomere, at rest and during muscle contraction, is shown
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Chapter 4
Fig. 4.12. The organization of sarcomeres into myofibrils and myofibres.
in Fig. 4.12. The entire sarcomere extends from Z-line to Z-line. The region
occupying the outer portions of the sarcomere, near the Z-lines, is
composed solely of the actin molecule and is called the I-band. The central
portion of the sarcomere contains the myosin molecule and is called the
A-band. The portion of the A-band that contains only myosin is called
the H-zone. The dark centre of the H-zone is known as the M-band and it
consists solely of the dense, central portion of the myosin molecule.
Muscle, Skeletal System and Adipose Tissue
73
Sarcomeric proteins: actin, myosin and titin
Myosin is the most abundant protein in muscle cells. It forms the thick filament of the sarcomere. The myosin molecule (∼220 kDa) is a dimer that consists of a long, rod-like tail composed of two intertwining heavy myosin
molecules that form an α helix, and a globular head (Fig. 4.13). Two myosin
light chains, which have regulatory functions, are associated with the neck
region of the heavy myosin chains. The myosin tail provides an anchor that
maintains the position of the myosin heavy chain (MHC). The myosin tail is
attached to the globular head domain of myosin via a hinged neck region.
The neck region allows the head of the myosin chain to ‘contract’, or
flex, much like your wrist allows your hand to flex, moving up and down
Fig. 4.13. Myofibrillar molecules.
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Chapter 4
without moving your forearm. The globular head of myosin contains binding sites for interaction with the thin actin filaments of the sarcomere. The
myosin head also contains an ATP-binding site that binds and hydrolyses
ATP by its ATPase enzymatic activity. Myosin head ATPase is activated by
ATP binding and degrades ATP to ADP, providing energy for the myosin
heads to flex and move along the actin filaments during muscle contraction
cycle. The golf club-shaped myosin molecules aggregate to form bundles of
tail-to-tail polymers of many myosin molecules that form a feathered
appearance, with the myosin heads projecting from the sides of the aggregate. These bundles of myosin molecules form the thick filaments of skeletal
muscle.
Actin, the second most abundant protein in muscle cells, forms the thin
filament of the sarcomere. The actin molecule consists of two strands of protein twisted upon one another, to form a rope-like, α-helical structure (Fig.
4.13). The long actin macromolecule is formed by a chain of multiple globular actin monomers, each of about 40 kDa in size. Actin contains the binding
site for the myosin head. Two tropomyosin molecules twist around the
length of the exterior of the actin molecule, residing in the groove formed
between the two actin polymers. When the muscle is at rest, tropomyosin
covers the myosin-binding site on actin. Another sarcomeric protein, troponin, binds to discrete sites on the tropomyosin molecule that occur at
every seven actin monomers. Troponin consists of three subunits. One binds
calcium during contraction (Tn-C), another is responsible for attaching troponin to tropomyosin (Tn-T) and the third, Tn-I, inhibits the interaction of
actin and myosin. When calcium binds to the Tn-C subunit of troponin, troponin undergoes a conformational change. As a result, troponin pulls
tropomyosin away from the actin–myosin-binding site on the actin molecule, exposing the myosin-binding sites on the actin molecule. Myosin can
now bind to this site, flex and initiate muscle contraction. By pulling the
actin molecules, immobilized at the Z-lines, toward the centre of the sarcomere, the sarcomeres are shortened, resulting in muscle contraction.
Another minor sarcomeric protein is titin, an elastic filament that connects the actin molecules to the borders of the sarcomere, the Z-discs. Titin is
the largest known protein, composed of a long single chain of more than
27,000 amino acids that stretch for 1.5 µm. The titin molecule has a molecular weight of about 3 million Da. Titin is important in maintaining the passive tension of muscles at rest, may act as a molecular template for sarcomere
organization and assists in the maintenance of the characteristic, highly
organized structure of the sarcomere.
Myofibres and muscle contraction
Mature muscle fibres, or myofibres, are characterized by the presence of high
proportions of mitochondria, which provide the energy, via oxidative phosphorylation of glucose, that is needed for muscle contraction.
Mitochondria may account for more than 20% of the muscle cell volume in
Muscle, Skeletal System and Adipose Tissue
75
highly oxidative muscles. Much of the remainder of the muscle fibre consists
of the insoluble protein molecules that are responsible for muscle contraction. Muscle cells secrete very little protein and this is reflected at the myofibre level by the low amount of endoplasmic reticulum within the cell. Most
proteins are synthesized by ribosomes within the cytoplasm of the myofibre.
The cytoplasm of the myofibre also contains energy reserves used for ATP
generation that powers muscle contraction and cell metabolism. There are
abundant glycogen granules near the sarcoplasmic reticulum and between
myofibrils of the muscle cells. Lipid vacuoles are also present in cells that
rely on oxidative metabolism.
Contraction of skeletal muscle is regulated by nerve impulses from
motor neurones that form contacts, or synapses, with the muscle cells. Each
muscle fibre is innervated by a motor neurone and the muscle fibre contracts
in response to the release of the neurotransmitter, acetylcholine, by the motor
neurones. A motor unit consists of a motor neurone and the muscle fibres
that it innervates. In general, a single neurone will innervate about 150 muscle fibres. The plasma membrane of the myofibre is called the sarcolemma
and invaginations of the sarcolemma form the transverse, or T-tubule structure
that penetrates deep into the muscle fibre (Fig. 4.14). The T-tubule extension
Fig. 4.14. Cross-section through a mature muscle cell.
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Chapter 4
of the sarcolemma provides a conduit for the neural activation signal into the
cell’s interior. This results in a more rapid response to stimulation than
would be permitted by simple diffusion of molecules from the cell surface.
Another specialized membrane within the muscle fibre, the sarcoplasmic
reticulum, acts as a connection between the T-tubules and the contractile
myofilaments. Calcium stored in the sarcoplasmic reticulum is released in
response to nerve impulses from the T-tubules and is required for muscle
contraction. The sarcoplasmic reticulum removes calcium from the myofilaments during muscle relaxation. Within the muscle, contraction is mediated
by the myofibrils, which contain the contractile apparatus of the myofibre.
Muscle contraction is initiated by the release of acetylcholine into the
neuromuscular junction. This results in the depolarization of the sarcolemma and this depolarization spreads to the interior of the myofibre via
the intracellular T-tubule system. The membrane depolarization induces
movement of calcium (Ca2+) from the sarcoplasmic reticulum into the cytoplasm (Table 4.1). Calcium binds to the troponin C subunit, stimulating a
conformational change in troponin. This results in the movement of
tropomyosin away from myosin-binding sites on the actin molecule resulting in the exposure of the actin–myosin-binding sites on actin, allowing the
formation of actin–myosin cross-bridges. The activated ATPase enzyme on
the myosin head converts ATP to ADP and phosphate. The enzymatic conversion of ATP to ADP increases the affinity of myosin for actin and the
energy produced is used to power the rapid conformational change in
myosin around its hinge points, resulting in sliding of the attached thin actin
filament towards the centre of the sarcomere. This results in a shortening of
the sarcomere and muscle contraction. At the end of a contraction cycle, calcium is actively transported back into the sarcoplasmic reticulum, where it
is no longer available to troponin. Tropomyosin then shields the
actin–myosin-binding sites and the ATPase in the myosin head becomes
inactive.
Table 4.1. The contraction cycle of skeletal muscle.
1.
Cytosolic Ca2+ is increased
2.
Troponin C binds Ca2+, uncovers tropomyosin-shielded myosin-binding sites on actin
3. ATP bound to myosin head reduces the strength of actin–myosin binding, and the
actin–myosin cross-bridge is broken
4. The unbound myosin head moves to a new actin-binding site and ATP is hydrolysed to
ADP
5.
The myosin head, with attached ADP, binds to a new actin site with high affinity
6.
The actin–myosin cross-bridge generates
7. Myosin undergoes a rapid conformational change around its hinge points and ADP is
released
8.
The thin actin filament is pulled toward the M-line of the sarcomere
Muscle, Skeletal System and Adipose Tissue
77
A single flexion of all actin–myosin contacts results in a muscle shortening of about 1%. As muscle contraction generally shortens muscles to about
30% of their resting length, the overall contraction is due to multiple attachment–release cycles between the actin and myosin molecules. The serial
arrangement of the sarcomeres also contributes to the overall shortening, as
the formation of the long myofilament composed of multiple repeating sarcomeric units allows the myofilament to act as a single unit, with the summation of single sarcomere shortenings, resulting in a longer overall
shortening of the muscle. The contraction cycle continues as long as ATP and
calcium are present. In the absence of ATP, the myosin head has a very high
affinity for actin and is not released. This is the cause of post-mortem muscle shortening and the rigor mortis of muscle, which occurs after death.
Skeletal Muscle Fibre Types
Not all muscle fibres are equivalent. We are all familiar with the distinct dark
and white meat of domestic poultry. The most basic classification of muscle
fibres is based on this gross observation and was formalized in the 19th century.
Dark meat has a higher proportion of red, slow twitch muscles, capable of sustained contraction. They are darker red than white muscle because of their
abundance of myoglobin, an oxygen-carrying protein in muscle, and their
highly vascularized nature. Red muscle fibres are involved in sustained, longterm activities such as standing and maintenance of posture. They are common
in leg and back muscles. Although generally classified as slow twitch muscles,
many red muscle fibres are fast twitch muscles. Large muscles that depend
upon glycolysis for their energy have muscle fibre diameters that are generally
larger than oxidative-type fibres. White muscle fibres, on the other hand, are
considered fast twitch muscles, capable of quick reactions and using oxidative
processes for energy production. Fast twitch muscles are used for sprinting, fine
motor control and quick reflexive actions, have lower turnover rates and are
more energy efficient. The gross classification of muscles into red or white types
offers a simplified version of the muscle types. In reality, muscles contain a
genetically determined, variable proportion of fast and slow twitch fibres. There
is an almost continuous gradation of morphological, contractile and metabolic
properties associated with muscles, but physiological and biochemical analysis
of muscles allows us to classify muscle types into very few categories.
Physiological analyses of muscles provide ways to classify muscle fibres
according to their functional characteristics. The contraction velocity, maximum contraction force and sustainability of that force, or fatigue resistance
can be measured in experimental settings. These properties allow the
characterization of the functional differences between muscles, but are difficult to obtain and require specialized equipment and training. These contractile characteristics are also reflected at the cellular and biochemical level,
where differences in contraction velocities and relative oxidative vs glycolytic enzyme activities can be revealed by measuring the relative activities
of enzymes involved in these processes. Characterization of muscle fibre
types on the basis of their oxidative potential is done by the use of
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Chapter 4
histochemical techniques. Staining of thin frozen sections of muscle with
enzyme-specific reagents can be correlated with the physiological characteristics of a particular muscle. The use of these stains can reveal the proportion
of fast and slow twitch fibres and the amount of oxidative vs non-oxidative
muscle fibres in a particular muscle. Three muscle enzymes are used as
markers to distinguish the characteristics of muscle fibres: myofibrillar
ATPase, succinate dehydrogenase (SDH) and α-glycerophosphate dehydrogenase (αGPDH). Myofibrillar, or myosin, ATPase activity is proportional to
the contraction velocity of a specific fibre and is used to distinguish between
fast and slow twitch fibres. SDH is a mitochondrial enzyme that catalyses
the conversion of succinate into fumarate during the Krebs, or citric acid
cycle. Its presence is elevated in mitochondrial-rich cells and indicates a high
level of oxidative metabolism. The last enzyme used in fibre typing, αGPDH,
is located in the cytoplasm and is a measure of the glycolytic, or anaerobic,
metabolism of the cell. The use of these histochemical stains can be used to
classify muscle fibres into fast or slow, oxidative or non-oxidative, and glycolytic or non-glycolytic. Using these three stains provides eight (23) different possible fibre types, but more than 95% of muscle fibres can be
categorized into only three major groups. These three muscle types are:
(i) fast glycolytic (FG) fibres with high levels of αGPDH and ATPase; (ii) fast
oxidative-glycolytic (FOG) fibres containing high levels of all three markers;
and (iii) slow oxidative (SO) fibres having high levels of SDH, but low levels
of ATPase and αGPDH (Table 4.2).
A more recent muscle typing method uses immunohistochemical (IHC)
methods to distinguish between different isoforms of the MHC. Using monoclonal antibodies directed toward specific sites on the MHC and comparing
results with fibres stained for the histochemical markers, four different fibre
types could be distinguished, one correlating with the slow twitch fibre type
and three with the fast twitch fibre types. Type 1 MHC fibres are found in
slow twitch muscles and cardiac muscle, type 2A MHC is in most FOG muscles. Types 2B and 2X are typical of FG muscle fibres and are fairly rare,
found only in extraocular, laryngeal and jaw muscles. Fibre type 2X is classified as a muscle fibre type that displays properties intermediate between
those of 2A and 2B. The 2X fibre types represent a small population of skeletal muscle fibres that do not readily fit into the other categories (Table 4.2).
Table 4.2. Skeletal muscle fibre types.
Twitch/metabolism
MHC type
ATPase
SDH
GPDH
Slow oxidative (SO)
Type 1
+
++++
+
Fast oxidative-glycolytic (FOG)
Type 2A
++++
++++
++++
Fast glycolytic (FG)
Type 2B
++++
+
++++
Fast intermediate (FG)
Type 2X
++++
+
++++
Muscle, Skeletal System and Adipose Tissue
79
Skeletal muscle protein turnover
While the number and size of skeletal muscle cells are important factors in
the determination of skeletal muscle growth and ultimate size, the rates of
skeletal muscle protein turnover are also essential for muscle growth.
Skeletal muscle protein levels result from a balance between protein synthesis and protein degradation. Different rates of skeletal muscle growth are
dependent upon regulation of muscle cell protein degradation to a greater
extent than the rates of protein synthesis. Protein accumulation in muscle
cells occurs in an environment in which degradation is inhibited and synthesized proteins can accumulate in the cell. In addition, the contractile
myofibrillar proteins have a finite lifespan and, like any cellular component,
must be degraded and replaced by newly synthesized proteins. Intracellular
enzymes called calpains play a prominent role in maintaining the balance
between muscle protein degradation and synthesis.
Calpains are calcium-dependent proteolytic enzymes found in all vertebrate cells studied and are especially important in the regulation of skeletal
muscle protein degradation. Two forms of these enzymes are present in muscle cells, distinguished by the concentrations of calcium required to activate
them. The m-calpains require 400 to 800 µM Ca2+ for activation, while the µcalpains are activated by lower levels of calcium, 3 to 5 µM. Binding to other
proteins called calpastatins inactivates the calpains. Calpains degrade specific muscle proteins, such as titin, desmin and troponin T as well as other
minor skeletal muscle proteins. Myosin is degraded slowly and incompletely by calpains and actin is unaffected by these proteases. The effects of
calpains on the degradation of minor proteins of the sarcomere results in the
loss of Z-discs from the sarcomere. This is due in part to the cleavage of the
N-terminal (Z-disc) end of the titin molecule that connects actin to the
Z-line. Calpains do not completely degrade proteins into small polypeptides
and amino acids. Calpain degradation results in the generation of large
polypeptide chains that are then degraded to amino acids by lysosomal proteases, the cathepsins. Calpains also degrade certain cytoplasmic enzymes
such as most protein kinases and phosphatases, leading to speculation that
calpains may have indirect regulatory functions within the cell, as kinases
and phosphatases activate and inactivate a variety of intracellular proteins.
While concentrations of calpains within the cell remain relatively constant, calpastatin levels fluctuate with physiological states. For example, in
animals treated with β-agonists, muscle protein hypertrophy is stimulated
while levels of calpastatin increase several fold. This is believed to inhibit calpain proteolysis and allow the accumulation of muscle protein. Likewise,
calpastatin activity is increased in specific muscles that undergo hypertrophy as a result of the Callipyge gene in sheep. These animals have a 30% to
50% increase in the muscle mass of skeletal muscles in the hind limb and
back regions. Calpastatin activity is increased specifically in these muscles,
but not in muscles that are unaffected by the Callipyge gene. Again, the
accumulation of protein in hypertrophied muscles is believed to result from
reduced protein degradation in the muscle cell as calpain activity is reduced
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by elevated calpastatin activity. Calpains are important in the post-mortem
tenderization of meat. Ninety-five per cent of the muscle protein proteolysis
that occurs in the first 1 to 2 weeks after slaughter is due to activation of
skeletal muscle calpains upon death of the animal.
The formation of multinucleated skeletal muscle cells
In biology, we now know many things that we take for granted. For example, we have known for many years that skeletal muscle cells were multinucleated. However, the mechanism by which these cells became
multinucleated was not known. Were the nuclei the result of replication of
nuclei within a single cell without cell division, a process called endoreduplication, or did the multiple nuclei result from fusion of many mononucleated precursor cells? In the mid-1960s, Beatrice Mintz set out to answer this
question using a sophisticated approach.
This study used mice from two different strains, one with a black fur
coat and one white. These strains carry different forms (isozymes) of the
enzyme isocitrate dehydrogenase (IDH). IDH consists of two protein chains
that are unique to the particular isozyme. The chemical differences in the
IDH isozymes result in different mobilities in an electric field, and the
isozymes could be distinguished from each other after separation by electrophoresis. Critical to the design of this study is the ability of these protein
chains to combine with one another in the cytoplasm to form hybrids of the
isozyme. These hybrid IDH molecules consist of a protein chain from each
of the isozymes. When subjected to electrophoresis, hybrid molecules
migrate a distance that is intermediate to that of the original IDH isozymes.
Thus, if the isozymes were from several genetically different nuclei, as
expected in the fusion model, an intermediate hybrid form (a/b) of the
enzyme would be present (Fig. 4.15). If the multinucleated cells were
derived from a single nucleus by endoreduplication, cells would contain
only one type of IDH gene and only the original forms (a/a or b/b) of the
isozymes would be present.
Drs Mintz and Baker created a new, mosaic animal by combining the
blastomeres of early, undifferentiated embryos from each of the strains.
These chimeric embryos, which the authors called tetraparental or
allophenic mice, were then transferred into recipient dams and allowed to
come to full term. Successful allophenic mice were phenotypically distinguished from the original black or white strains by their patched black and
white coat colour. Skeletal muscle and liver (used as a control, mononucleated tissue) were collected for electrophoretic analysis of isozyme patterns.
Isozyme patterns were compared with conventional reproductive crosses
between the strains of mice. As shown in Fig. 4.16, skeletal muscle from conventional matings and from the allophenic mice contained the original
isozymes as well as the intermediate form. This combination of all three
combinations of IDH isozymes was expected if multinucleation resulted
from fusion of myoblasts containing different IDH alleles. The IDH isozyme
Muscle, Skeletal System and Adipose Tissue
81
Fig. 4.15. Two models that may account for the formation of multinucleated myotubes.
Fig. 4.16. Pattern of IDH enzyme activity observed after electrophoretic separation of tissues
isolated from skeletal muscle (SM) or liver (L): 1, IDH strain b, SM or L; 2, IDH strain a, SM or
L; 3, pooled tissue of strain a and strain b, SM or L; 4, F1 cross of IDH strain a plus IDH strain
b, SM or L; 5, allophenic (chimeric) strain a/b SM; 5, allophenic strain a/b, L. The arrow on the
right indicates the direction of electrophoresis. Source: Mintz and Baker (1967).
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pattern of mononucleated liver tissue from allophenic mice, however, displayed only the original isozyme patterns. This was the expected result from
a tissue that contained the isozyme alleles in separate cells. Liver tissue from
conventional matings, in which genes from both parents are present in a single nucleus, also contained the intermediate hybrid form of the IDH enzyme.
Thus, it was established that multinucleated skeletal muscle cells were
derived from fusion of many precursor cells and not from the replication of
a single nucleus within a precursor cell.
Adipose Tissue Growth and Development
We are all familiar with adipose tissue, or fat, both on a personal level and
on a nutritional level. Diet-induced obesity and the accompanying Type II
diabetes are common in the human populations of Western Europe and the
USA and are prominent health concerns in these countries. Likewise, when
we consume meat products, we savour the flavours imparted by the adipose
tissue, which is part of all meats. In animal production, fat is an important
component of edible meat, but is an expensive addition. The deposition of
appropriate quantities of fat in an animal destined for consumption is an
important component of the costs of animal production and the suitability of
the product for human consumption. The length of time taken to fatten food
animals is a major cost for producers. On the other hand, the accumulation of
intramuscular fat increases the quality and value of the meat product.
Fat is an energy-dense tissue. When consumed, the lipids in fat produce
roughly 9 kcal/g, while those of protein and carbohydrate produce about
4 kcal/g. This means, of course, that the biological synthesis of lipids also
requires over twice the amount of food energy than carbohydrate or protein.
Thus, the deposition of fat is a costly enterprise, both metabolically and economically. A great deal of the subcutaneous fat deposited in food animals is
wasted, as it is trimmed from carcasses and discarded or used as the basis to
make products of lesser value. It has been estimated that the USA produces
over 2 billion kilograms of excess lipid each year from the production of food
animals. Because of the economic importance of adipose tissue and the negative economic impact of excess subcutaneous adipose tissue deposition,
knowledge of the developmental and cellular processes of fat formation is
essential to understand the regulation of fat deposition and to eventually
improve farm animal production efficiency.
Brown adipose tissue
Mammalian adipose tissue exists in two forms, white adipose tissue, the
most common form, and brown adipose tissue. Brown adipose tissue is
found in various locations, which are species-dependent. While the primary
role of white adipose tissue is energy storage, brown adipose tissue is
adapted for energy use and heat production. Brown adipose tissue is richly
Muscle, Skeletal System and Adipose Tissue
83
vascularized and contains abundant mitochondria for energy production
from lipid and glucose catabolism. Brown adipose tissue is also characterized by the presence of high levels of the protein, uncoupling protein-1
(UCP1), which functions to uncouple mitochondrial oxidative phosphorylation from ATP production. This results in the release of energy in the form
of heat. This is known as non-shivering thermogenesis and brown adipose
tissue is the sole source. Heat produced by non-shivering thermogenesis is
essential to maintain homeostatic body temperature in neonatal animals and
in animals emerging from hibernation. This ensures neonatal survival and
survival in cold climates. At birth, with the exception of subcutaneous fat,
essentially all adipose tissue in cattle and sheep can be considered to be
brown adipose tissue. Brown adipose tissue in newborn sheep and cattle is
replaced by white adipose tissue within the first week of neonatal life. This
occurs without a change in adipocyte cell numbers and is believed to result
from transdifferentiation of brown adipocytes into white adipocytes. In the
adult, brown adipose tissue responds to environmental and nutritional cues.
For example, in adult rats, brown adipose tissue depots undergo enlargement via hyperplasia in response to cold acclimation and to increased food
intake. Activation of brown adipose tissue thermogenesis is controlled by
norepinephrine release from the sympathetic nervous system (SNS).
White adipose tissue
White adipose tissue is the tissue we generally think of as fat and is the subject of this section. White adipose tissue is the major storage site for energy
in the form of lipid, when dietary energy sources exceed energy use by the
animal. This energy reserve can be used during times of food scarcity. In
addition, subcutaneous white adipose tissue provides a layer of insulation
that prevents heat loss in cold environments. Many internal organs are surrounded by a layer of visceral fat that provides a mechanical cushion to protect organs against physical trauma. Since the discovery in 1994 that white
adipose tissue secretes the hormone leptin, adipose tissue has been recognized as an endocrine tissue. Adipose tissue is no longer considered as just
a relatively passive storage reservoir, but is involved in regulation of
appetite, reproduction and energy homeostasis. The endocrine function of
adipose tissue will be discussed in subsequent chapters.
Fat is a diffuse organ that is present in several discrete sites, or depots,
throughout the body. Fat depots are present in many visceral sites in association with the internal organs. For example, fat is found as perirenal, omental, cardiac and mesenteric depots. Fat also develops in association with
internal reproductive organs such as the uterus (parametrial) and the testes
(epididymal). Subcutaneous fat, located between the muscle and skin of
animals, is the most easily identified and morphologically visible of the fat
depots. In addition, fat infiltrates muscles, where it is found as intermuscular or seam fat, between large muscle groups and within muscles as intramuscular fat. Developmentally, the visceral fat depots are formed first,
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Chapter 4
followed by the subcutaneous fat depot, and finally, in adulthood, intramuscular fat is formed. Depot sites are constant in mammals, but proportions vary with species. For example, subcutaneous fat is much more
prominent in pigs than in beef cattle and relatively sparse in dairy cattle.
Likewise, beef cattle have much more abdominal fat than dairy cattle.
White adipose tissue consists primarily of lipid (76–94%) and, like bone,
is classified as a connective tissue. Nearly 90% to 99% of lipid in the
adipocyte consists of triacylglycerols, with small amounts of free fatty acids,
phospholipids, mono- and diacylglycerols and cholesterol. Adipose tissue is
composed of two distinct components: the lipid-containing fat cells or
adipocytes and the non-lipid component, the stromovascular cells. The stromovascular portion of adipose tissue consists of a variety of blood cells,
fibroblasts, endothelial cells, pericytes and adipocyte precursor cells. The
stromovascular cell fraction of adipose tissue provides an important reservoir of stem cells for production of new adipocytes. Adipocyte precursor
cells can be isolated from the stromovascular cell fraction of the neonatal,
adult and even senescent animal. These preadipocytes can be grown in vitro
and are important sources of cells to study the regulation of adipocyte
differentiation.
Adipose tissue development
Adipocytes develop from embryonic mesenchymal cells beginning in mid to
late gestation in mammals. The specific time of initiation of adipocyte formation varies with the depot site and the species studied. In the fetus, adipogenesis occurs in close association with vascular development. In the
adult, fat is highly vascularized and each adult adipocyte is in contact with
at least one capillary. Adipocyte precursor cells aggregate as fat cell clusters
in fat depot sites that surround existing capillary beds. These fat primordia
increase in size and number during fetal development and undergo rapid
expansion in the neonatal animal. The increase in neonatal adipose mass is
the result of hyperplasia of precursor cells combined with hypertrophy of
existing adipocytes. Most of the body’s fat reserves develop postnatally. At
birth, the bodies of farm animals contain roughly 1–4% fat, while at maturity, these animals can have as much as 40% of their body weight as fat.
In the adult, adipocyte precursor cells are derived either from the circulation, adjacent tissues or the stromovascular cells of the adipose tissue. It is
not clear if the stem cell is a fibroblast, macrophage or blood vessel pericyte.
In any case, stem cell replication produces adipoblasts, which are morphologically indistinguishable from other cells, but are committed to the
adipocyte lineage. Adipoblasts replicate and differentiate into preadipocytes
(Fig. 4.17). Again, newly formed preadipocytes are not morphologically distinct from other cells, but as preadipocytes differentiate, they are characterized by the accumulation of small lipid droplets and the appearance of
lipogenic enzymes. An early marker of differentiation is lipoprotein lipase
(LPL), whose function is to assist in the accumulation of lipids in the cell.
Muscle, Skeletal System and Adipose Tissue
85
Fig. 4.17. Differentiation of adipocytes.
After lipid filling and cellular enlargement the cell assumes the typical
‘signet ring’ morphology of the mature adipocyte. This results from the accumulation of a large lipid droplet within the cell that forces the cytoplasm and
nucleus of the adipocyte to the periphery. The cell is now capable of expression of the full complement of enzymes that catalyse lipogenesis, such as
fatty acid synthase (FAS), pyruvate carboxylase and acetyl-CoA carboxylase
(ACC).
Adipose tissue growth: hyperplasia vs hypertrophy
How do adult animals accumulate large amounts of fat? Up until the mid1970s, it was thought that a specific number of fat cells was present at birth
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Chapter 4
and the number of adipocytes did not change during the lifetime of the animal. This was supported by the observation that mature adipocytes were
incapable of mitosis. It is now known that fat accretion in the adult is a function
not only of lipid accumulation and an increase in adipocyte size, or hypertrophy, but also as a result of an increase in fat cell numbers by hyperplasia.
In the adolescent animal, nutrition and exercise affect adipocyte numbers. These changes in adipocyte cell numbers persist into adulthood. In the
adult, early observations suggested that total cell number in adipose tissue
depots were constant and unaffected by typical regulators of cell growth
such as nutrition, exercise and hormones. These observations gave rise to an
early theory of adipose tissue accumulation called the critical period theory
that divided the growth of adipose tissue into two phases. The first is the
developmentally early, sensitive, pre-adult phase, characterized by marked
adipoblast hyperplasia and sensitivity to external stimuli. This is followed
by the adult phase, characterized by stable, unchanging adipocyte cell numbers. In the adult phase, adipocyte growth was thought to occur solely by the
accumulation of lipids, or fat cell hypertrophy, the enlargement of fat cells.
This early work was criticized on several points, many of them apparent in
hindsight, after the development of more sophisticated methods to study
adipocytes. For example, early studies used inadequate methods to count
adipose cells; rats and hamsters, which are now known to be unique in their
absence of adult cell division, were used as model organisms; and depots,
which are not representative of overall fat growth, were studied. For example, most of the early studies used rodent epididymal fat as an epididymal
fat depot model. While this depot is abundant and readily accessible in
rodents, epididymal fat is not representative of adipose tissue growth seen
in subcutaneous and visceral fat.
It is now known that the number of adipocytes is not fixed at birth and
that adult adipose depots increase in size by mechanisms which involve
both hyperplasia and hypertrophy. Faust and co-workers postulated the
maximum fat cell size theory in 1978. This theory posits that adipocyte numbers initially stabilize at a certain level before maturity. During this phase,
cell numbers are constant but adipose cells accumulate lipid and cell size
continues to increase. When a certain ‘maximum fat cell size’ is reached in
early adulthood, new fat cells are recruited from stem cells or adipoblast precursors (Fig. 4.18). Evidence now exists that when adipocytes reach a certain
maximal size, they secrete growth factors and other paracrine agents that
Fig. 4.18. Changes in fat cell size and
numbers during postnatal life. Adapted from
Johnson and Francendese (1985).
Muscle, Skeletal System and Adipose Tissue
87
stimulate the growth and differentiation of adipocyte stem cells in the stromovascular fraction of adipose tissue. This will be discussed in a later chapter that deals with the regulation of adipocyte development. Recruitment of
new cells results in an increase in fat cell numbers. Addition of these new
undifferentiated cells to the fat depot reduces the average fat cell size in the
adipose depot. This cycle of adipocyte hypertrophy and hyperplasia can be
repeated several times during adulthood and senescence, with the volume of
fat cells reaching a maximum again and again, followed by the recruitment
of new fat cell precursors for addition to the adipocyte depot. This theory
provides a more flexible, realistic picture of fat cell development in the adult
and has generated new interest in the study of fat cell morphogenesis and its
regulation.
Lipogenesis and Lipolysis
Adipose tissue plays an essential role in the storage and release of energy by
sequestering and mobilizing fat, or triacylglycerols. The adipocyte is the primary cell that stores fats, and fats are constantly degraded and resynthesized
in the adipocyte. The main storage form of fat is triacylglycerol, a macromolecule consisting of a glycerol backbone esterified to three long chains of fatty
acids. Triacylglycerols may be supplied to adipocytes from the liver and the
intestine in the form of serum lipoproteins. These complexes of fat and protein appear in the circulation as chylomicrons, from intestinal absorption, or
as high, low or very low-density lipoproteins (abbreviated HDL, LDL and
VLDL, respectively) derived from the liver.
LPL, an enzyme synthesized by many tissues and localized on the
endothelial cell surface of capillaries, catalyses the removal of fatty acids
from the lipoproteins. After the fatty acids enter the cell, they are reconverted to triacylglycerols by adipocytes. Triacylglycerols may also be synthesized from circulating free (i.e. non-esterified) fatty acids that circulate in
the bloodstream bound to albumin. After their uptake by the cell they are
esterified to glycerol to form triacylglycerols (Figs. 4.19 and 4.20). These
mechanisms of triacylglycerol formation are common in many omnivores or
carnivores that consume diets relatively high in lipids. In animals that consume small amounts of lipids (as well as omnivores and carnivores) longchain fatty acids may also be synthesized de novo by the process called
lipogenesis. Lipogenesis per se involves the synthesis of fatty acids from
small, unrelated precursor molecules that are derived from rumenal fermentation or glycolysis of sugars. The conjugation of fatty acids to glycerol to
form triacylglycerols is a process separate from lipogenesis.
In humans and birds, lipogenesis occurs primarily in the liver. In ruminants and pigs, the adipocyte is the main site for lipogenesis. Fatty acids are
synthesized from the two-carbon precursors, acetate or lactate, or from glucose after it has been converted to pyruvate by glycolysis (Fig. 4.21). While
glucose is the primary precursor of fatty acids in non-ruminants, microbial
catabolism of glucose in the rumen of ruminants results in the production of
88
Chapter 4
O
HO
C CH2 CH2 CH2 CH2 CH2 CH2 CH2
CH=CH CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2
Oleic acid
O
HO
C CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2
Palmitic acid
(A) Examples of fatty acids
H
H C OH
H C OH
+
3 Stearic acid
H C OH
H
H
H C O
C (CH2)16 CH3
H C O
O
C (CH2)16 CH3
H C O
O
C (CH2)16 CH3
H
O
Triacylglycerol
(B) Formation of triacylglycerols
Fig. 4.19. Fatty acids and the formation of triacylglycerols.
pyruvate and acetate. Acetate is the major precursor for lipogenesis in these
animals. The synthesis of long-chain fatty acids occurs in the cytoplasm by
the repeated addition of two carbons to a ‘primer’ molecule, which is usually acetyl-coenzyme A (acetyl-CoA).
ACC is the enzyme that catalyses the initial, rate-limiting step of lipogenesis. This results in the conversion of acetyl-CoA to malonyl-CoA. MalonylCoA then acts as the donor of two-carbon acetyl groups to the growing fatty
acid chain. Increased circulating insulin concentrations, in response to
dietary energy, induce ACC gene transcription and translation resulting in
an increase in ACC and increased rates of lipogenesis and fatty acid formation. The insulin counter-regulatory hormones, glucagon, epinephrine and
norepinephrine, inhibit the activity of ACC. This occurs in times of fat mobilization during times of food shortage or in stressful situations.
After the formation of malonyl-CoA, FAS catalyses the remainder of the
lipogenic cycle. FAS is a large macromolecular complex (MW ∼500,000) con-
Muscle, Skeletal System and Adipose Tissue
89
Fig. 4.20. Structure and nomenclature of fatty acids.
sisting of two identical protein subunits, arranged head-to-tail. Each subunit
contains seven different catalytic activities and the molecular arrangement
of the homodimers allows FAS to catalyse the simultaneous synthesis of two
fatty acid chains. Although FAS is not a rate-limiting enzyme for lipogenesis, it is essential for the chain elongation of fatty acids. The overall equation
for the synthesis of palmitic acid (a 16-carbon fatty acid) is:
Acetyl-CoA + 7malonyl-CoA + 14NADPH + 14H+
→ Palmitic acid + 7CO2 + 8CoA + 14NADP+ + 6H2O
Palmitic acid is the primary product of lipogenesis in adipose tissue. It
is converted to the 18-carbon stearic acid (C-18) in the endoplasmic reticulum by the enzyme fatty acid elongase. Stearoyl-CoA desaturase (SCD or ∆9
desaturase) converts a portion of stearic acid into oleic acid, the 18-carbon
non-saturated form of stearic acid, by the insertion of a double bond
between carbons 9 and 10. The presence of oleic acid, which has a lower
melting point than stearic acid, ensures the fluidity of lipids.
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Chapter 4
Fig. 4.21. Lipid metabolism.
The long-chain fatty acids that are synthesized during lipogenesis are
then esterified to a glycerol backbone. Concentrations of non-esterified fatty
acids are very low in the body and the formation of triacylglycerols provides
a non-toxic storage form for lipids. In ruminants, non-saturated fats derived
from the diet are quickly hydrogenated to saturated fats in the rumen before
intestinal absorption and, as a result, ruminant fatty acids are, in general,
more saturated than those of non-ruminants.
Mobilization of fat stores occurs when dietary energy intake is reduced
or when the animal is subjected to stress. An overall view of lipid metabolism is given in Fig. 4.21. The process of lipolysis is initiated by the sequential release of fatty acids from the glycerol backbone of triglycerols. The
initial lipolytic event is catalysed by hormone-sensitive lipase, an enzyme
that is activated by the hormone messenger, cAMP. The primary hormonal
inducers of lipolysis in mammals are the catecholamines, epinephrine and
norepinephrine. In birds, glucagon is the main inducer of hormone-sensitive
lipase. The activity of hormone-sensitive lipase determines the amount of
fatty acids released by adipocytes. Thus, this enzyme is the major determinant of blood lipid levels. Long-chain fatty acids released from adipose tis-
Muscle, Skeletal System and Adipose Tissue
91
Fig. 4.22. The β-oxidation cycle of fatty acids.
sue into the systemic circulation are only slightly soluble in water. Therefore,
they circulate bound to albumin in the bloodstream.
Fatty acids from the circulation are taken up by cells to resynthesize fats
or to generate energy by fatty acid catabolism. Degradation of fatty acids
occurs in the mitochondria by a process known as β-oxidation. Cleavage of
the long-chain fatty acids occurs at the carboxyl end of the fatty acid between
carbon-2 (the α-carbon) and carbon-3 (the β-carbon), hence the name
β-oxidation (Fig. 4.22). This occurs after binding of fatty acids to coenzyme A
in the cytoplasm. The activated fatty acid (acyl-CoA) is then transported into
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Chapter 4
the mitochondria via the carnitine shuttle system. In the mitochondria, the
long-chain fatty acids are sequentially degraded to two-carbon acetyl-CoA
residues. The complete oxidation of a long-chain fatty acid requires several
trips through the β-oxidation cycle. For example, eight cycles are required
for the oxidation of stearic acid, an 18-carbon fatty acid. The acetate molecules, in the form of acetyl-CoA, then enter the citric acid cycle and the respiratory chain to undergo final oxidation to CO2 with a concomitant
production of ATP. The energy balance for the complete oxidation of
palmitic acid (16:0) is shown in Fig. 4.23. A total of 106 ATP are produced per
molecule of palmitic acid, for a free energy gain of 3300 kJ/mol. The amount
of energy from the degradation of fatty acids is much greater than from the
degradation of carbohydrates (32 ATP per molecule of glucose) or from proteins, even when the different sizes of the molecules is considered. This
demonstrates the superiority of the use of fat as an energy storage molecule.
Fig. 4.23. Summary of the energy produced from fatty acid degradation.
References and Further Reading
Ailhaud, G., Grimaldi, P. and Negrel, R.
(1992) Cellular and molecular aspects of
adipose tissue development. Annual
Review of Nutrition 12, 207–233.
Allen, R.E., Merkel, R.A. and Young, R.B.
(1979) Cellular aspects of muscle growth:
myogenic cell proliferation. Journal of
Animal Science 49, 115–127.
Brook, C.G.D. (1972) Evidence for a sensitive
period in adipose cell replication in man.
Lancet ii, 624.
Drackley, J.K. (2000) Lipid metabolism. In:
D’Mello, J.P.F. (ed.) Farm Animal
Metabolism
and
Nutrition.
CAB
International,
Wallingford,
UK,
pp. 97–119.
Faust, I.M., Johnson, P.R., Stern, J.J. and
Hirsch, J. (1978) Diet-induced adipocyte
number increase in adult rats: a new
model of obesity. American Journal of
Physiology 235, E279.
Muscle, Skeletal System and Adipose Tissue
Goll, D.E., Thompson, V.F., Taylor, R.G. and
Ouali, A. (1998) The calpain system and
skeletal muscle growth. Canadian Journal
of Animal Science 78, 503–512.
Johnson, P.R. and Francendese, A.A. (1985)
Cellular regulation of adipose tissue
growth. Journal of Animal Science
61(Suppl. 2), 57–75.
Lieber, R.L. (2002) Skeletal Muscle Structure,
Function and Plasticity. Lippincott,
Williams & Wilkins, Philadelphia,
Pennsylvania, 369 pp.
Mersmann, H.J. (1991) Regulation of adipose tissue metabolism and accretion in
mammals raised for meat production.
In: Pearson, A.M. and Dutson, T.R. (eds)
Advances in Meat Research, Vol. 7.
Elsevier Applied Science, New York,
pp. 135–168.
93
Mintz, B. and Baker, W.B. (1967) Normal
mammalian muscle differentiation and
gene control of isocitrate dehydrogenase
synthesis. Proceedings of the National
Academy of Sciences USA 58, 592–598.
Nakagawa, N., Kinosaki, M., Yamaguchi, K.,
Shima, N., Yasuda, H., Yano, K.,
Morinaga, T. and Higashio, K. (1998)
RANK is the essential signaling receptor
for osteoclast differentiation factor in
osteoclastogenesis. Biochemical and
Biophysical Research Communications 253,
395–400.
Takahashi, N., Udagawa, N., Takami, M. and
Suda, T. (2002) Cells of bone, osteoclast
generation. In: Bilezikian, J.P., Raisz, L.G.
and Rodan, G.A. (eds) Principles of Bone
Biology, Vol. 1, 2nd edn. Academic Press,
New York, pp. 109–126.
5
Growth Hormone
and Insulin-like Growth Factors
Evans and Simpson first discovered a pituitary factor that enhances animal
growth in the 1920s when a bovine pituitary extract was shown to promote
growth in rats. Based on this observation, the activity was termed ‘growth
hormone’ (GH). Later studies in the following decade demonstrated that the
anterior pituitary extracts also had metabolic effects; reducing carcass fat
and increasing milk yield in treated animals. Isolation of GH from anterior
pituitary glands was accomplished in 1945 and its activity in promoting
growth and lactation was correlated to the original crude anterior pituitary
gland extracts.
Advances in molecular biology and genetic engineering in the late 1970s
and early 1980s allowed the use of recombinant DNA to produce large
amounts of bovine GH (bGH) from bacterial plasmids containing the bGH
gene. The availability of large quantities of bGH provided enough bGH, and
later porcine GH (pGH), to be used in studies of the effects of GH in large
domestic animals. The availability of GH for studies in large farm animals has
provided a wealth of information about the effects of GH on physiological
processes in ruminant and non-ruminant animals. These basic studies, in
turn, have led to the commercialization of the use of GH to promote lactation
in dairy cattle.
It is now known that many of the effects of GH are indirect, as one of the
major target organs of GH is the liver. Under GH stimulation, the liver synthesizes and secretes IGF-I, which acts as a hormone to induce many of the
effects that were originally believed to be direct effects of GH on other target
tissues such as muscle, bone and adipose tissue. The aim of this chapter is to
first examine the biochemistry, mechanism of action and metabolic effects of
GH and then to outline the IGF system and its physiological effects. Finally,
studies on the effects of GH and IGF-I treatment of farm animals will be
discussed.
The GH Molecule
GH, also called somatotropin (ST), is synthesized and secreted by the somatotrope cells of the anterior pituitary gland. It is a single-chain protein with
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Growth Hormone and Insulin-like Growth Factors
95
a molecular weight of about 22,000. In most species, GH consists of
191 amino acids and contains two disulphide bonds that maintain its threedimensional structure. The amino acid sequence of GH is similar to prolactin
(PRL) and placental lactogen (PL) (Fig. 5.1). PRL is a hormone that is primarily involved in the induction of milk synthesis in mammals. PL is synthesized in the placenta during pregnancy and is thought to be a regulator
of fetal glucose supply, maternal mammogenesis, and in some species, fetal
growth. Due to this overlapping amino acid homology, PRL, PL and GH
have overlapping biological actions.
Fig. 5.1. Structures of growth hormone and prolactin. Both have two cysteines that form
intramolecular covalent disulphide bonds, 190 to 200 amino acids and regions of identical
amino acid sequences.
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Chapter 5
Control of GH secretion
Circulating concentrations of GH vary widely. The secretion of GH from
the anterior pituitary is pulsatile, and GH concentrations in plasma occur
as irregular spikes of concentration followed by very low levels of GH.
The specific pattern of GH release is related to the species, sex and age of
the animal. Spikes in GH concentrations occur roughly six to eight times
during a 24 h period. For this reason, the accurate measurement of blood
GH concentrations is difficult and time-consuming. Single blood samples,
sufficient for quantitation of other hormones, are inadequate to measure
GH and lead to samples with wide variations of GH concentrations.
Sequential blood samples, taken at 15 or 20 min intervals over a 6 to 8 h
period are required to establish a useful record of overall GH concentrations. When GH concentrations are measured, data are accumulated on
overall, mean GH concentration, frequency of GH pulses and high and
low GH levels. The actions of GH are believed to be dependent upon not
only overall concentrations, but also maximum GH pulse levels and the
frequency of GH secretory pulses. Enhanced effects on growth promotion
are seen when GH is administered in multiple, small doses rather than as
a large, single, daily injection or a continuous infusion. This is an impractical treatment in animal production systems, but reflects the endogenous
rhythms and complex mechanisms that regulate animal growth in the
intact system.
The release of GH from the anterior pituitary gland is influenced by several physiological factors, including neurotransmitters, hypothalamic peptides and circulating metabolites. Specific CNS neurotransmitters that affect
the hypothalamus such as serotonin precursors, GABA and dopamine alter
GH release. Reduced levels of blood glucose (hypoglycaemia) and concomitant elevations of blood insulin induce GH release. High concentrations of
circulating amino acids, especially arginine, induce GH release, while elevated levels of circulating IGF-I, free fatty acids and obesity inhibit GH
secretion. GH levels are increased during the physiological states of sleep,
stress, exercise and starvation.
As with all anterior pituitary hormones, the secretion of GH is controlled
by the hypothalamus (Fig. 5.2). The hypothalamus produces peptides that
stimulate and inhibit the release of GH from the anterior pituitary.
Traditionally, two hypothalamic peptides, one stimulating and one inhibiting GH release, were believed to be the only controls of GH release. It is now
known that additional hypothalamic peptides are involved in the control of
GH release from the anterior pituitary.
The release of GH from the anterior pituitary is induced by hypothalamic
GHRH. GHRH is a 44-amino acid peptide synthesized by cells in the arcuate
(ARC) nucleus of the hypothalamus (Fig. 5.3). GHRH is secreted from neurosecretory nerve terminals in the median eminence and transported to the anterior pituitary gland by the hypophyseal portal system. GHRH binds to a
G-linked protein receptor in somatotropes, stimulating an increase in cAMP
and activating GH release via the transcription factor, pit-1.
Growth Hormone and Insulin-like Growth Factors
97
Fig. 5.2. Hypothalamic control and negative feedback loops for the regulation of GH secretion.
Somatostatin (SS) or GHRIH is produced in the periventricular nucleus
of the hypothalamus. It is a tetradecapeptide (14 amino acids) that is found
in many tissues outside the hypothalamus. This includes the CNS and delta
cells of endocrine pancreas and gut. As the name implies, SS inhibits the
release of GH from the anterior pituitary by reducing cAMP concentrations
within the somatotropes. In addition, it inhibits TRH-induced release of TSH
(but not TRH-induction of PRL release). SS also acts as an inhibitor of several other hormones and metabolites including PTH, calcitonin, gastric HCl,
acetylcholine and catecholamine neurotransmitters.
Although TRH is the primary hypothalamic peptide that induces pituitary TSH release, it also induces GH release in birds, cattle and rats. TRH is
a tripeptide [(pyro)glu-his-pro-amide] produced in the paraventricular
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Chapter 5
Fig 5.3. Amino acid sequences of growth hormone releasing hormone, somatostatin and ghrelin.
nucleus (PVN). The amino acid sequence of TRH is identical in porcine,
ovine, bovine and human species. In addition, TRH induces PRL release in
essentially all vertebrates studied.
The traditional concept of the regulation of GH release from the anterior
pituitary primarily by hypothalamic stimulating (GHRH) and inhibiting (SS)
factors has been modified by the discovery of an additional regulatory
polypeptide. In the 1970s and 1980s, studies were carried out with synthetic
polypeptides and non-peptides that induce GH release (GH secretagogues),
with a goal of providing human diagnostic and treatment tools. These studies suggested that the synthetic secretagogues acted through a receptor distinct from the known GHRH receptor. This ‘orphan’ receptor, which had no
known natural ligand, was called the GH secretagogue (GHS) receptor.
Using a ‘reverse pharmacology’ approach, in which extracts from several
organs were tested for their GHS receptor activity, it was found that the
stomach was the major source of the natural GHS receptor ligand. This led
to the isolation of a peptide by Japanese scientists in 1999 that was named
ghrelin. The term ghrelin is derived from the Proto-Indo-European root
‘ghre’, for the word ‘growth’. The suffix ‘relin’ means ‘releasing substances’.
Ghrelin is a 28-amino acid polypeptide that was the first naturally occurring polypeptide to contain an octanoylated amino acid (ser-3). Ghrelin regulates energy expenditure, appetite as well as the GH axis. Ghrelin is
produced by the pituitary and hypothalamus as well as mucosal enteroendocrine cells of the stomach. Ghrelin mRNA has also been found in
descending concentrations in the small and large intestines, and in the pancreas, liver and kidney. Its secretion is induced by fasting and reduced by
feeding. Ghrelin receptors are G-protein-linked receptors and are found in
the hypothalamus, pituitary and various areas of the brain. Ghrelin acts
Growth Hormone and Insulin-like Growth Factors
99
through the hypothalamus and the pituitary to induce pituitary release of
GH. The ghrelin mRNA and peptide in the anterior pituitary are increased by
GHRH infusion and ghrelin may have a local autocrine or paracrine effect on
stimulating GH release. In addition to the regulatory effects on GH, ghrelin
has several direct physiological effects related to energy intake and utilization. Acting via neuropeptide Y (NPY) in the hypothalamus, ghrelin
increases appetite, food intake and body weight gain. In addition, ghrelin
has local effects in the gastrointestinal tract, where it stimulates gastric acid
secretion and gastric motility. Thus, ghrelin provides an endocrine link
between the stomach, hypothalamus and pituitary and coordinates the
neuroendocrine regulation of energy balance with the GH/IGF-I system.
GH receptors
The GH receptor was the first member of the cytokine/haematopoietin
receptor superfamily to be identified and cloned from human and rabbit
liver in 1987. This receptor has now been cloned from laboratory rodents,
cattle, sheep, swine and chickens. The GH receptors are classified as class I
cytokine/haematopoietin receptors and also include the receptors for PRL,
PL, leptin, erythropoietin, interleukins and the colony-stimulating factors
(CSF) for immune cells. Class II cytokine receptors include those for the
interferon-α/β, interferon-γ and interleukin-10.
There are three types of GH/PRL family of receptors that bind the GHand PRL-related hormones to varying degrees and specificity in different
species. The GH receptor is termed a somatogenic (growth-inducing) receptor. GH receptors are widespread and are found in most of the tissues of the
body. The liver is an abundant source of the GH receptor, where it mediates
the induction of IGF synthesis and secretion. GH receptors are also found in
bone, cartilage, adipose tissue and skeletal muscle. The bGH receptor binds
bovine, ovine and human GH with equal affinities, with a lower affinity for
PRL and PL. The lactogenic receptor is found in many tissues, but is abundant in the mammary gland and liver. In the liver, the PRL receptor concentrations are present in higher concentrations in females than males, and are
induced by oestrogens. This receptor binds PRL and PL with equal affinities
that are higher than that of GH. The PL receptor of the placenta binds PL
with greater affinity than PRL and PRL better than GH. The cross-reactivity
of GH, PRL and PL with each other’s receptors means that these hormones
are capable of inducing growth and lactation. Although the primary function
of GH is growth regulation and it is generally a weak promoter of milk synthesis, it is a strong inducer of lactogenesis in some species such as cattle.
Likewise, the lactogenic effects of PRL and PL predominate, while these hormones have weak growth-promoting actions.
The GH receptor is a 120–130 kDa highly glycosylated protein,
which consists of a single polypeptide chain (∼620 amino acids, 70 kDa),
containing a single transmembrane domain, without intrinsic kinase
activity (Fig. 5.4). There are ten tyrosine (Y) residues in the cytoplasmic
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Chapter 5
Fig. 5.4. The GH receptor, showing asparagine (N) sites for glycosylation, cysteines (C) that
form intramolecular disulphide bonds, tyrosines available for phosphorylation and the Box 1
site for JAK2 binding.
domain of the GH receptor, some of which are phosphorylated by JAK.
Multiple cysteines (C), forming disulphide bonds, are present in the
extracellular domain. This domain is highly glycosylated on the numerous extracellular asparagines (N). After binding to GH, this receptor
monomer forms a dimer with another monomeric GH receptor, binds
JAK2 on the proline-rich Box 1 site of the cytoplasmic domain and activates the STAT transcription factors by tyrosine phosphorylation. The
activation and signal transduction of the GH receptor via JAK has been
discussed in Chapter 3.
Interestingly, the extracellular, ligand-binding domain of GH receptor is
found in the circulation of all animals examined, including cattle, sheep,
swine, horses, goats, cats and dogs. This truncated receptor acts as a GHbinding protein (GHBP) and is composed of roughly 246 amino acids that are
identical to the extracellular domain of the GH receptor. It is produced primarily in the liver, where it is co-expressed with the GH receptor. The formation of
the GHBP results from proteolytic cleavage of the GH receptor near the trans-
Growth Hormone and Insulin-like Growth Factors
101
membrane site in rabbits and humans. In rodents, alternate splicing of the GH
gene produces the GHBP. This soluble GH-binding entity is glycosylated,
devoid of the transmembrane domain and the cytoplasmic transducer of the
complete receptor. Circulating concentrations of GHBP are higher in females
than males, increase with age and are correlated with body mass. It has been
proposed that GHBP is an indicator of GH status and sensitivity. In addition,
GHBP may regulate the activity of circulating GH by sequestering it, preventing its degradation and, thus, increasing its half-life in the circulation.
Metabolic actions of GH
GH is an anabolic hormone that affects most tissues. The effects of GH can
be broadly divided into those which alter metabolism and those which promote growth of bone and skeletal muscle. These actions are, of course, overlapping, but it is convenient to examine the metabolic and growth effects
separately. The metabolic effects of GH are wide-ranging and affect all three
metabolic fuels: carbohydrates, proteins and fat (Table 5.1).
Table 5.1. Effects of growth hormone on metabolism of major nutrients.
Nutrient
GH effect
Proteins
Enhanced amino acid uptake and utilization (muscle)
Enhanced protein synthesis, accretion (muscle)
Reduced amino acid oxidation, catabolism (liver)
Reduced blood urea nitrogen
Reduced urinary nitrogen excretion
Carbohydrates
Increased glucose uptake (muscle)
Reduced glucose uptake and oxidation (adipose)
Increased gluconeogenesis/glycogenolysis (liver)
Increased glucose release (liver)
Reduced glucose clearance and catabolism
Increased blood glucose
Reduced insulin responsiveness
Lipids
Reduced lipogenesis (chronic, positive energy balance)
Reduced insulin sensitivity
Enhanced lipolysis (acute, negative energy balance)
Reduced expression and activity of lipogenic enzymes
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Chapter 5
GH effects are species-specific. Humans will respond only to GH of
human or primate origins. Before the production of GH from recombinant
DNA, only GH from cadaver pituitaries could be used to treat GH-deficient
humans and the use of purified GH to enhance animal production was
unthinkable. GH from animals ‘lower’ on the evolutionary scale does not
affect humans. Domestic animals respond best to GH from their own
species. This is because of the diversity of protein hormones, which display
different amino acid content between species. For example, the amino acid
similarity between ovine and bGH is essentially identical (99.5%), while
pGH is about 90% similar to bGH. In contrast, human GH has an amino acid
similarity to bGH of only 68%. These differences in amino acid sequence
between species are manifested in the binding to hepatic receptors from different species. Thus human GH is only one-tenth as effective in binding to
the bGH receptor as is bGH. For this reason, bovine, ovine and pGH have no
effects on humans.
Major target organs for GH are the liver, skeletal muscle, bone/cartilage
and fat, but GH receptors are found in virtually all tissues. It is now known
that most of GH effects on growth are mediated by IGF-I, which is induced
at the local target tissue by GH. Within the tissues, IGFs, induced by GH, act
primarily on a local level in autocrine and paracrine mechanisms. The effects
of GH are often difficult to demonstrate in vitro, in the absence of IGF-I. This
and other evidence suggests that many of the traditional or classical effects
of GH seen when it is injected into animals are indirect and mediated by the
IGF system (Fig. 5.5). The relationship between GH and the IGFs will be
more fully elaborated after the following discussion of the effects of GH on
nutrient metabolism.
The anabolic effects of GH on protein metabolism are seen at many levels. These effects can be detected in the whole animal and in individual
organs, although many of these effects are probably indirect and due to
direct effects of IGF-I. In general, when animals are treated with GH, this
induces an overall positive nitrogen balance in the body. This is reflected by
a reduction in circulating amino acids, decreased blood urea nitrogen concentrations and reduced urinary nitrogen excretion. This nitrogen-conserving
effect of GH provides amino acids for increased muscle hypertrophy and
muscle protein accretion in growing animals. In lactating animals, these
amino acids are directed towards milk protein synthesis. GH treatment stimulates skeletal muscle and mammary gland amino acid uptake, reduces
hepatic amino acid catabolism and increases protein synthesis in vivo. These
effects are not seen in isolated muscle or mammary cells, suggesting that
these effects of GH are indirect and mediated by IGF-I.
GH also plays a role in regulating carbohydrate metabolism. Along with
glucagon, the glucocorticoids and the catecholamines, GH acts as an insulin
counter-regulatory hormone to mobilize tissue stores of glucose to increase
blood glucose levels. Treatment of lactating cows with GH reduces whole
body glucose oxidation and increases the irreversible loss of glucose. GH has
anti-insulin effects, inhibiting the actions of insulin on adipose glucose
uptake. GH also blocks the inhibitory effects of insulin on hepatic gluconeo-
Growth Hormone and Insulin-like Growth Factors
103
Fig. 5.5. The direct and indirect effects of GH.
genesis. In lactating cows, de novo synthesis of glucose via gluconeogenesis
provides virtually all of the energy needed for milk synthesis. Liver stores of
glycogen in these animals are inadequate to meet the needs for the energy
required for milk synthesis. GH is also considered to be a diabetogenic hormone, increasing blood glucose and, in response to elevated glucose, circulating insulin concentrations. When given over long periods of time, GH can
induce permanent diabetes, probably due to pancreatic islet cell ‘burnout’ in
response to chronically elevated glucose. Hence, many of the effects of GH
that result in an elevation of blood glucose are due to an inhibition of insulininduced glucose utilization by the body, not to a direct stimulation of gluconeogenesis by GH.
GH treatment reduces lipid deposition. GH has effects on both lipogenesis and lipolysis, with the major effect on reducing lipogenesis, rather than
increasing lipolysis. Although early studies reported lipolytic effects of GH,
these have not been seen with highly purified recombinant GH used in
experiments conducted since the 1990s. Effects of GH on lipid metabolism
are chronic, seen over long periods of treatment in vivo and in vitro. GH
effects on lipid metabolism are largely indirect, as GH acts by inhibiting
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Chapter 5
insulin effects on adipose tissue glucose uptake via the cellular glucose
transport protein, GLUT4, and subsequent glucose use for lipogenesis. GH
treatment reduces the gene expression and activity of the key, rate-limiting
lipogenic enzymes, FAS and ACC. In well-fed animals, lipolytic effects of
GH are minor and GH treatment does not induce an increase in circulating
free fatty acids and glycerol, products of lipolysis. However, if animals are in
a negative energy balance, treatment with GH can induce lipolysis. This
effect is transient and indirect. This is seen at the beginning of GH treatments
of lactating dairy cattle, growing cattle and pigs, and is thought to be due to
an increased lipolytic response to catecholamines, adrenal medullary hormones released in response to stress.
Excess and inadequate GH: giants and dwarfs
Excess or inadequate concentrations of GH lead to growth and metabolic
anomalies with phenotypic and metabolic consequences. These are well
characterized in humans and are representative of effects seen in animals
with GH anomalies. The effects of excess GH depend upon the developmental stage in which the animal is exposed to GH. Excess GH secretion is
usually seen as a result of an adenoma, an anterior pituitary gland tumour.
When excessive GH is present prior to puberty, gigantism results. In this condition, the epiphyses of the long bones fuse at a later age than normal, resulting in excessive long bone growth and height. Constant exposure to GH in
the growing animal also affects the visceral organs, resulting in splanchnomegaly, the enlargement of the liver, kidneys and the gastrointestinal
tract. Skin and skeletal muscles also are hypertrophic under the influence of
GH. Affected individuals suffer from diabetes due to the constant elevation
of glucose, and subsequently insulin, under the influence of GH.
When adults are exposed to excess GH, a condition known as acromegaly
results. This is characterized by growth of bones at the sites of existing cartilage and results in the enlargement of the extremities (feet, hands, nose, jaw)
and a resultant ‘coarsening’ of features. These characteristics are due to the
lateral overgrowth of bones after the epiphyses have fused. Other features of
acromegaly include thyroid goitre, osteoarthritis, carpal tunnel syndrome
and reproductive disorders.
Insufficient GH, as a result of hypopituitarism, leads to dwarfism.
Hypopituitarism can result from a variety of causes, ranging from pituitary
tumours, trauma to the pituitary, radiation exposure or genetics. Other than
specific genetic defects in GH secretion or insensitivity, these hypopituitary
syndromes are relatively non-specific and affect GH as well as the
gonadotrophic hormones LH and FSH. As a result, most hypopituitary
dwarfs are reproductively sterile and have infantile body proportions, with
large heads and lack of secondary sex characteristics. More recently a syndrome called Laron dwarfism has been identified in humans. Affected individuals have normal levels of GH, but do not have the GH receptor, and are
thus GH-resistant. A line of miniature Brahman cattle has been found to be
Growth Hormone and Insulin-like Growth Factors
105
the result of a mutation in the GH receptor, similar to the Laron dwarf syndrome in humans. As in the human syndrome, this animal is GH-insensitive
and displays elevated circulating GH and reduced serum levels of IGF-I.
Reduced growth rate and proportionally reduced adult body size have also
been found in humans with mutations in the GHRH receptor, in which low
circulating levels of GH and IGF-I are present as well as normal body proportions. In general, individuals affected with the classic hypopituitary
dwarfism syndrome are phenotypically different from the small people
called midgets. The latter condition is hereditary rather than hormonal and
results in a smaller adult body form that is proportional to the conventional
adult form. Midgets are fertile and possess all the attributes of normal-sized
adults.
IGFs or Somatomedins
IGFs are small peptides which mediate many of the effects of GH. These
growth factors play multiple roles in animal growth and development, acting as circulating hormones as well as growth factors that are produced in
most, if not all, tissues that act via local autocrine and paracrine pathways.
Their actions encompass insulin-like effects on metabolism, GH-like effects
on animal growth and effects on cell division and differentiation in tissues
derived from embryonic mesoderm.
Discovery of the IGFs
In 1957, Salmon and Daughaday published the first evidence for serum factors which had direct effects on cartilage growth. These scientists were
studying the effects of serum on the incorporation of radioactive sulphur
into chondroitin sulphate of rat cartilage in vitro, with a focus on GH effects.
They found that serum from normal rats stimulated sulphate incorporation
while serum from hypophysectomized rats had no effect. This suggested
that GH was stimulating cartilage growth. However, when GH was added
to the hypophysectomized rat serum there was no effect on sulphate incorporation. When injected into hypophysectomized rats, GH treatment
induced a positive growth response. These observations suggested that GH
did not have a direct effect on cartilage growth and that the effects of GH
were mediated by a circulating factor not produced by the pituitary. This
was the first evidence for the existence of a sulphation factor which mediated
GH effects. Dr Daughaday later named this somatomedin, a factor which
mediates the effects of ST, or GH. The somatomedin fraction was later separated into two different sub-fractions, somatomedin C, whose serum concentration was GH-dependent, and somatomedin A, which was not.
The initial observations of Daughaday’s group languished until 1963,
when Swiss scientists (Froesch, Rinderknecht, Humbel) were examining the
paradoxical findings that serum had insulin-like effects on increasing
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Chapter 5
glucose uptake in skeletal muscle and fat cells which were much greater than
expected from insulin alone. These conclusions were due to the observation
that antibodies to insulin did not suppress these insulin effects. This group
called this activity non-suppressible insulin-like activity (NSILA). After isolation
and separation of the NSILA activity into two distinct fractions, this activity
was renamed IGF-I and IGF-II.
In 1972, Pierson and Temin were investigating low molecular weight
serum factors which stimulated cell growth in vitro. They succeeded in isolating mitogenic peptides from serum with estimated molecular weights less
than 10 kDa. As a result of the mitogenic effects of these peptides, Pierson
and Temin called this activity multiplication-stimulating activity (MSA).
Further studies by Temin’s group showed that the liver was the source of
MSA. It is now known that the original MSA is equivalent to IGF-II.
These studies established the IGFs as versatile molecules which display
the dual characteristics of serum-borne endocrine hormones and mitosisinducing growth factors. The IGFs have effects on cartilage growth, stimulate mitosis in many cells and have both insulin-like and GH-like effects.
These studies led to the formulation of the somatomedin hypothesis
(Fig. 5.6). This hypothesis states that the effects of GH on target tissues are not
direct, but are mediated by IGFs synthesized and secreted by the liver under
the control of GH. The promulgation of this hypothesis, in the early 1980s,
altered fundamentally the field of endocrinology. The discovery and characterization of the biology of the IGFs demonstrated, for the first time, that a
traditional organ of metabolism, the liver, also acted as an endocrine organ
that secreted IGFs into the circulation. It also relegated GH to the status of an
inducing factor that had few, if any, direct effects on organs other than the
Fig. 5.6. The somatomedin hypothesis of GH action.
Growth Hormone and Insulin-like Growth Factors
107
liver. In the ensuing years, it has been shown that GH has both direct and
indirect effects on some tissues and that the role of endocrine IGF is less than
that of IGF produced locally under the stimulation of GH. Modifications and
extensions of the somatomedin hypothesis will be discussed later in this
chapter.
IGF genes and molecules
Although the mature IGF peptides contain at most 70 amino acids and could
be coded for by 210 bases, the IGF genes are very large and complex. The
IGF-I gene consists of at least six exons separated by five introns and covers
more than 80 kb of DNA. The IGF-II gene consists of nine exons and spans
about 30 kb of DNA. Several different IGF mRNA species arise from transcription of the genes due to alternative splicing, polyadenylation and multiple promoter sites. Some transcripts are specific to different organs and
tissues and may play important roles in tissue development and embryonic
growth and differentiation.
The two types of IGF, IGF-I and IGF-II, are distinguished by their biochemical and biological characteristics. IGF-I is considered to be the primary
biologically active IGF in adult mammals and is secreted in an endocrine
fashion by the liver in response to GH. IGF-I was originally dubbed
somatomedin C by Daughaday and is produced not only by the liver, but
also by many other tissues. IGF-I has a basic isoelectric point (pI) of about 7.8
and consists of 70 amino acids in all mammals studied. IGF-I is thought to
mediate many of the effects of GH and is important for late fetal growth and
initiating early postnatal growth. IGF-II (identical to somatomedin A and
MSA) is a polypeptide with a neutral pI of about 7.0 and consists of 66 or
67 amino acids in mammals. It is secreted independently of GH and is thought
to be important in the regulation of early fetal growth.
The IGFs have a molecular structure that is very similar to proinsulin,
the precursor to insulin (Fig. 5.7). Both IGF-I and IGF-II consist of a single
polypeptide chain with molecular weights of about 7500 Da. Like proinsulin,
the IGF polypeptides contain two disulphide bonds to maintain the tertiary structure and consist of three functional domains (A, B, C). When the
inactive proinsulin is converted to active insulin, the C domain (C-peptide) is
enzymatically removed, resulting in an active insulin molecule with two
peptide subunits held together by disulphide bonds. Unlike insulin, the
C-peptide is not cleaved in IGFs. The A and B domains of the IGFs are about
45% identical to insulin. The C domain consists of 31 to 35 amino acids in
proinsulin, 12 in IGF-I and eight in IGF-II. The sequence of the IGF-I and
IGF-II molecules is highly conserved between different mammalian species.
The IGF-I peptide is identical in humans, swine and cattle, with a single
amino acid change in ovine IGF-I, three for rats and four in mice. Bovine and
human IGF-II differ from one another by three amino acids, while rat and
mouse IGF-II differ from the human molecule by four and six amino acids,
respectively.
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Chapter 5
Fig. 5.7. The primary structures of (A) porcine proinsulin and (B) human IGF-I. The amino acids
in black circles of the IGF-I structure are identical in both human insulin and IGF-I.
IGF receptors
The effects of the IGFs are mediated by three receptors which bind IGFs, the
insulin receptor, the Type I IGF receptor and the Type II IGF receptor
(Fig. 5.8). The overlapping biological effects of the IGFs and insulin are
reflected not only by the overall gross similarity of these molecules, but also
by the overlapping molecular identity and cross-reactivity of ligand binding
by the insulin and IGF-I receptors. The insulin and Type I IGF receptors are
very similar in overall structure and their amino acid sequences are about
30% identical. These large molecules (∼450 kDa) are heterotetramers, consisting of two identical extracellular ligand-binding α subunits and two β
subunits containing the transmembrane and cytoplasmic domains. The cytoplasmic β subunits of the insulin and Type I IGF receptors are TK which
catalyse the phosphorylation of intracellular second messengers. These
receptors were discussed in detail in Chapter 3.
In contrast, the third receptor for IGFs, the Type II IGF receptor, consists
of a single glycoprotein chain, with a molecular weight of ∼250 kDa. This
molecule is also known as the cation-independent mannose-6-phosphate
(M-6-P) receptor. The plasma membrane form of this receptor consists primarily
of an extracellular domain that consists of 15 repeats of approximately 147
amino acids. The extracellular domain has two binding sites for M-6-P and a
single IGF-II binding site. The Type II receptor is embedded in the plasma
Growth Hormone and Insulin-like Growth Factors
109
Fig. 5.8. Characteristics of the IGF and insulin receptors.
membrane by its transmembrane domain, but has only a truncated cytoplasmic tail. It is the primary cell surface binding entity for IGF-II. IGF-I, but not
insulin, also binds slightly to this receptor. The Type II IGF receptor has no
known kinase activity or associated second messenger system. For this reason,
it is not believed to function as a hormone receptor. It is located primarily
within the intracellular membranes of the cytoplasm where it binds lysosomal
enzymes via their M-6-P residues. The primary biological function of the IGFII/M-6-P receptor is believed to be in transporting hydrolytic enzymes from
the Golgi apparatus to the lysosomes, where they are involved in cellular
catabolism of specific substrates. In addition, the Type II IGF receptor is
involved in the clearance and catabolism of circulating IGF-II. The plasma
membrane form of this receptor is rapidly internalized and IGF-II bound to
the receptor is degraded in lysosomes. The internalization and breakdown of
IGF-II prevent development of excessive levels of IGF-II, which can be detrimental to the animal, especially during embryonic development. Knockout
mice in which the Type II receptor is deleted exhibit elevated IGF-II concentrations, enlarged viscera and rarely live beyond birth. These effects are
believed to be a result of exposure to excessive IGF-II, which results as a consequence of the absence of degradation by the Type II receptor. The biological
effects of IGF-II are mediated through the IGF-I receptor.
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Chapter 5
IGF-binding proteins
The adult, circulating concentrations of the IGFs are quite high for a hormone, generally above 100 ng/ml (13 nmol/l). Cellular responses to IGF-I
are induced at much lower levels, in the order of 10 to 20 ng/ml. The potential effects of excess serum concentrations of the IGFs are dampened by the
binding and sequestration of IGFs by serum binding proteins. Roughly 99%
of circulating IGFs are bound to binding proteins. In addition, many tissues
produce the IGFBPs locally. These binding proteins reversibly bind IGF-I
and IGF-II to high affinity, non-covalent binding sites. The affinity of IGFBP
for IGFs is 10 to 40 times higher than the IGF-I receptor, effectively removing IGFBP-bound IGF from interactions with its target tissue receptors.
There are six IGFBPs, numbered 1 to 6. Most of the IGFBPs have equal
affinities for IGF-I and IGF-II, but IGFBP-6 binds IGF-II with a 20- to 70-fold
higher affinity than IGF-I. The major serum IGFBPs were discovered when
IGF activity was isolated in the high molecular weight fractions after size
exclusion chromatography of serum. These early studies showed that a
majority of IGF activity was associated with the 40 and 150 kDa molecular
weight fractions, with 70–80% of the IGF activity in the high molecular
weight, 150 kDa fraction. This high molecular weight binding protein was
named IGFBP-3. GH stimulates the hepatic secretion of this glycoprotein.
IGFBP-3 consists of an acid-stable subunit (40–45 kDa), which binds IGF, and
an acid-labile subunit (85 kDa). The second major serum-binding protein is
IGFBP-2. This IGFBP accounted for the other 20% to 30% of bound serum
IGF, originally designated as the 40 kDa fraction. It is now known that
IGFBP-2 is a 31.3 kDa protein that is also secreted by the liver. The other
IGFBPs are single-chain proteins, of roughly 25 kDa. These include IGFBP-1,
a placental protein found mainly in fetal amniotic fluid. IGFBP-4 is widespread and found in serum, seminal plasma, fibroblasts and osteoblasts.
IGFBP-5 and IGFBP-6 are less abundant binding proteins found in serum
and tissues.
In general, circulating IGF bound to IGFBP is unavailable for receptor
binding and is not biologically active. The regulation of IGF actions is related
to the interactions of IGF with binding proteins in the serum and at the tissue level. The IGFBPs are thought to regulate IGF action in a number of
ways. As the binding is reversible and in chemical equilibrium with nonbound (free) IGF, the IGF–IGFBP complex provides a constant source of
additional IGF. As free IGF levels are reduced by IGF use and metabolism, it
is replaced by the release of IGF from the IGFBP. In addition, IGFBP-bound
IGF has a longer half-life than unbound, or free, IGF. Thus, binding to IGFBP
increases the circulating half-life of IGF, probably by reducing IGF degradation.
In contrast to the primarily inhibitory effects of IGFBP on IGF activity,
three IGFBPs increase the activity of IGFs: IGFBP-1, -3, and -5. This enhancement of IGF activity is attributed to different mechanisms. Phosphorylation
of the IGFBP-1 molecule increases its affinity for IGF by sixfold and the
lower affinity of dephosphorylated IGFBP-1 induces the release of bound
Growth Hormone and Insulin-like Growth Factors
111
IGF. This enhances the effect of IGF on DNA synthesis in some cells. This
increased IGF activity is thought to be due to a shift of the binding of IGF
from the lower affinity, dephosphorylated IGFBP to the higher affinity IGFI receptor. IGFBP-3 binds to cell surfaces while IGFBP-5 is associated with
the ECM. The binding to cell membranes and matrix proteins reduces the
affinity of IGFBP-3 and -5 for IGF, resulting in the release of IGFs, which
stimulate tissue receptors. Local production of IGFBP may also protect IGFs
from proteolytic degradation, provide a local tissue reservoir of IGF, and
present IGF to local tissue receptors. IGFBP-1 and -2 have an Arg-Gly-Asp
(RGD) sequence which binds to specific surface-bound integrin receptors. In
general, the actions of the IGFBP are likely to play a more pronounced regulatory role in the localized actions of IGF.
From a practical standpoint, the measurement of IGFs using competitive
binding methods, such as radioimmunoassay or radioreceptor assay, are
greatly influenced by the presence of the IGFBPs. Whether the biologically
relevant portion of the IGFs is the total (free and IGFBP-bound) IGF or the
free (non-bound) IGF, the measurement of IGFs cannot be accomplished in
the presence of the IGFBPs, due to the IGFBP competition for binding sites
on the immunoglobulins or receptors used for analysis. The IGFBPs can
be removed physically, using size exclusion chromatography to separate the
high molecular weight IGFBPs from the smaller IGFs. Analysis of the low
molecular weight fraction provides a measure of free IGF and is likely the
most relevant measure. This method is cumbersome, expensive and timeconsuming. The majority of reports of IGF concentrations use a chemical
denaturation method, by treatment of serum with acid or acid–ethanol. This
removes the IGFBPs and releases IGFs for analysis, providing a measure of
total IGF. As IGFBP types and affinities vary with species, developmental
stage (fetal, neonatal and adult) and physiological state (e.g. pregnant vs
non-pregnant), methods to remove these binding proteins must be validated
for each application.
Metabolic effects of the IGFs
Adult serum levels of IGF-I are very high for a hormone, about 100 to
150 ng/ml, while those of IGF-II are about threefold higher. The liver produces about 90% of the IGF-I in the circulation and, until recently, IGF-I was
thought to act in a traditional endocrine manner. High serum IGF-I levels act
in a negative feedback loop at the hypothalamus, inducing SS release and,
hence, reducing GH secretion. Additional IGF is produced in most, if not all,
tissues, where it acts in an autocrine and/or paracrine manner, affecting
local tissues without the intervention of the circulation. Recent data, discussed below, suggest that local tissue production of IGF-I is more important
in regulating animal growth than is endocrine IGF-I.
The IGFs have primary effects as mitogenic and differentiation-inducing
growth factors and secondarily as insulin-like factors. The mitogenic effects
of the IGFs are quite pronounced and the IGFs are active at low nanomolar
112
Chapter 5
concentrations. Treatment of cells with IGF stimulates RNA and DNA synthesis in many cells with a mesodermal origin. In addition, IGFs increase
amino acid uptake, inhibit protein degradation and stimulate protein synthesis in many tissues. In cartilage, IGFs increase collagen and proteoglycan
synthesis. In fat cells, the IGFs stimulate lipid synthesis and reduce lipolysis,
effects opposite to those of GH. Specific effects of the IGFs will be discussed
in the following chapters on muscle, fat and bone growth and development.
As we have seen, IGFs cross-react with the insulin receptor at higher
pharmacological concentrations. At higher concentrations, the IGFs can
have insulin-like effects. IGF-I is about 1/100 to 1/5 as potent as insulin in
inducing insulin-like effects, probably via interactions with the insulin
receptor. For example, high doses of IGF-I induce increased glucose transport
and oxidation by adipose tissue. In addition, at high concentrations, IGF-I can
induce transient hypoglycaemia, accompanied by increased glycogen synthesis in liver and muscle. Overall, these insulin-like effects of the IGFs
induce anabolic metabolism in target tissues, leading to a net increase of
glycogen, protein and lipid deposition. Interestingly, when present in pharmacological doses, insulin can act as a mitogen, a process presumably mediated
by the IGF-I receptor.
Effects of the IGFs on animal growth
The IGFs have many effects on animal growth. Early observations showed
that, in the growing animal, IGF levels increase with postnatal age. IGF-I
serum concentrations in the neonatal animal are relatively low, but gradually
increase during maturation, reach a peak at the time of puberty and then
gradually decline throughout adulthood. Several studies have shown that
serum levels of IGF-I are proportional to birth weight and to adult body
weight. In many species, including humans, livestock and companion animals, IGF-I concentrations are proportional to body size. In dogs, for example, IGF-I concentrations increase with breed sizes, from miniature to toy to
standard-sized poodles (Table 5.2).
Similar observations have been made in other species, including swine,
cattle and sheep. The relatively constant circulating levels of IGF-I, propor-
Table 5.2. Correlation of plasma IGF-I concentrations and adult body size of dogs.
Poodle breed
Weight (kg)
IGF-I (ng/ml)
Toy
3.0±0.2
16±4
Miniature
6.2±0.4
24±5
Standard
20.9±1.6
From Eigenmann et al. (1984).
96±15
Growth Hormone and Insulin-like Growth Factors
113
tional to GH concentrations, are much easier to measure than GH, and provide an excellent measure of the growth and GH status of animals. When
IGF-I is injected into hypophysectomized or normal animals, growth rates
are increased (Fig. 5.9). Transgenic IGF-I mice, which express excess IGF-I,
have higher growth rates than normal mice, but have a disproportionate
increase in the sizes of brain, pancreas, kidney, spleen and thymus. In general, IGF-I is not as potent as GH in growth induction and does not mimic all
effects of GH.
Fetal and early neonatal growth in most species is independent of GH.
Animals without a pituitary gland, GH or the GH receptor develop normally
and have expected birth weights. In the mammalian fetus, circulating concentrations of GH are relatively high, but the fetal liver has little or no GH
receptor, and IGF secretion is independent of GH. In the fetal sheep liver, for
example, GH receptors are undetectable, but the fetus has abundant receptors for PRL and low levels of PL receptor. Binding to hepatic GH receptors
increases rapidly in the newborn lamb, and as GH binding increases, there
is a proportional increase in postnatal serum IGF-I levels, suggesting that the
appearance of hepatic GH receptors is functional and mediates the induction
of IGF-I secretion that stimulates postnatal growth. In calves, there is no
detectable GH binding to liver receptors up to the second day postpartum.
Significant GH binding is not seen until 3 weeks postpartum, and increases
up until 12 weeks.
Fig. 5.9. Effects of GH and IGF injections on growth of hypophysectomized rats. (With
permission from Schoenle et al., 1985.)
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Chapter 5
The effects of removing the influence of IGF-I and IGF-II on fetal and
postnatal growth have been explored in ‘knockout’ mice that have had the
IGF-I or -II gene deleted. When the IGF-I gene is deleted, the majority of animals die immediately after birth due to the lack of skeletal muscle, and
hence diaphragm, development. Surviving animals are stunted, weighing
only 60% of normal birth weight and at adulthood are only 30% of the normal body weights. This suggests that IGF-I plays a major role in fetal as well
as postnatal growth. Treatment of growing, IGF-I-deficient mice with GH
does not improve growth, suggesting that the presence of IGF-I, either local
or endocrine, is required for the growth-promoting effects of GH. When the
IGF-II gene is deleted, most animals are viable, but are only 60% of normal
birth weight and adult body weight. Thus, the major role of IGF-II is during
fetal development, with minimal or no effects on postnatal development.
Most of the IGF-II effects during fetal development are indirect, due to
effects on placental growth. The IGF-II knockout mice have reductions in
placental weights that are proportional to the reduction of birth weights.
Recent studies have examined the role of endocrine vs paracrine and
autocrine effects of IGF-I on animal growth. In these experiments, the
hepatic IGF-I gene is selectively deleted. This removes the endocrine source
of IGF-I without affecting local tissue production of IGF-I. In these animals,
only extrahepatic, locally produced IGF-I is present. In the non-selective IGF
knockout mice described above, the IGF genes are inactive in all tissues and
throughout fetal, neonatal and adult development. The selective deletion of
hepatic IGF-I gene occurs in the late fetal stages of development. Hence, the
IGF-I effects on fetal growth are largely eliminated.
In the animals that lack the hepatic IGF-I gene, circulating IGF-I is
reduced by 75% or more, while GH concentrations are elevated fourfold.
This indicates that endocrine IGF-I is important in the negative feedback
control of GH secretion, as the lower circulating levels of IGF-I are believed
to relieve the normal GH inhibition by IGF-I. Surprisingly, the growth of
these animals, as reflected by femoral bone and body length and organ
mass, was not altered. In addition, sexual maturation and lactation were
normal. Messenger RNA concentrations for IGF-I in heart, skeletal muscle,
fat, spleen and kidney were unaltered, suggesting that IGF-I gene expression was not increased in the absence of circulating IGF-I or the presence
of excess GH. The lack of a GH effect on tissue levels of IGF-I mRNA in
these mice has been ascribed to the observation that GH is no longer
released in a pulsatile fashion, which may be needed for mRNA induction.
Likewise, there was no change in the concentrations of tissue IGF-I receptor mRNA.
In sum, these studies with targeted deletion of the liver IGF-I gene have
demonstrated that circulating endocrine IGF-I is not involved in the regulation of postnatal growth. Locally produced IGF-I is the major regulator of
postnatal growth. The control of local IGF-I synthesis and secretion is, in
many cases, dependent upon GH stimulation and GH can have direct as
well as indirect effects (through local IGF-I production) in stimulating the
growth of adipose tissue, bone and skeletal muscle.
Growth Hormone and Insulin-like Growth Factors
115
Modification of the somatomedin hypothesis
The original somatomedin hypothesis posited that most, if not all, effects of
GH were mediated by endocrine IGF, produced by the liver under GH stimulation. This hypothesis was formulated before it was shown, in 1980, that IGF
mRNA was present in virtually all fetal, neonatal and adult tissues. The validity of the somatomedin hypothesis was tested by Isaakson in 1982. To examine the direct effects of GH on longitudinal bone growth, in the absence of liver
production of IGF-I, GH was injected directly into the tibial growth plate of
hypophysectomized rats. This stimulated bone growth in the injected, but not
the uninjected tibia, suggesting that GH had a direct effect on bone growth
and that endocrine IGF from the liver was not involved. However, when antiserum to IGF-I was injected with GH (to inhibit the effects of local IGF-I) no
bone growth was observed. These studies showed that the GH effects on bone
growth were indeed indirect, but were not mediated by endocrine IGF-I.
Instead, GH stimulated the production of local, bone-derived IGF-I, which
acted in a paracrine or autocrine manner to induce tissue growth.
These observations, and similar ones by Green using adipose tissue, led
to the formulation of the dual effector hypothesis for GH/IGF-I effects on tissue growth and differentiation (Fig. 5.10). This hypothesis proposes that GH
Fig. 5.10. The dual effector hypothesis of GH and IGF-I action.
116
Chapter 5
stimulates the initial differentiation of adipocyte and chondrocyte precursor
cells. This results in two events: the development of IGF-I responsiveness
and the expression of the IGF-I gene. IGF-I, in turn, is secreted locally and it
acts to stimulate mitosis and clonal expansion of adipocytes and chondrocytes, acting in a local, autocrine or paracrine fashion. Amplified cell populations then undergo final maturation. This modification of the somatomedin
hypothesis accounts for the dual roles of GH and IGF-I on the growth and
differentiation of cartilage and adipose tissue. Together with the observations discussed earlier in mice with selective deletion of the hepatic IGF-I
gene, and the emphasis on autocrine and paracrine actions of IGF-I, the original somatomedin hypothesis must be reconsidered. With the exception of
the negative feedback control of GH secretion, the major effects of the IGFs
on growth and development are likely not to be endocrine in nature.
Although these effects are stimulated by treatment with either GH or the
IGFs, they are mediated by the IGFs in a local, tissue-specific manner via
autocrine and paracrine mechanisms (Fig. 5.11).
Effects of GH on Farm Animal Production
The observations that the GH/IGF-I system enhanced muscle deposition
and reduced fat content, coupled with the relatively new science of recombinant DNA for production of large quantities of these proteins, led to several trials of GH (called bST by Monsanto) on animal production systems
beginning in the early 1980s. Monsanto was the first company to put a large
Fig. 5.11. The modified somatomedin hypothesis. (After LeRoith et al., 2001.)
Growth Hormone and Insulin-like Growth Factors
117
effort and investment into the production and use of GH to enhance animal
production. As with the β-agonists, the major effects of GH in enhancing
farm animal production result from nutrient repartitioning, by directing
nutrient energy away from fat deposition and toward milk or muscle protein
synthesis.
The primary focus for the commercial use of GH in animal production is
in dairy cattle. The commercial use of bST in the USA began in 1994 and GH
is now used in more than 2 million dairy cattle in 25 countries. The European
Union prohibits its use. GH is given during the last 80% of lactation and
increases milk yield by 10–15%. GH has no effects on milk composition,
including protein, lactose, fat and minerals/vitamins. GH and IGF-I concentrations in milk are unaffected. As with all growth promotants, GH-treated
animals require increased nutrients, primarily in the form of protein and
amino acid supplements, which provide the needed energy and amino acid
precursors for increased milk production. The increase in milk production
efficiency offsets the added costs of feed supplementation.
The chemical instability of the GH molecule makes GH treatment of animals a problem. As GH is a protein, it is digested with other dietary proteins
in the gastrointestinal tract. This makes the oral administration of GH
impossible. Like most proteins, GH is susceptible to oxidation and denaturation, especially when in dilute aqueous solutions. Thus, injectable GH must
be kept refrigerated when not in use and, in early trials, daily injections of
GH were administered. Relatively long-lasting implants containing 500 mg
of GH have now been developed and marketed for use in dairy cattle, but
these must be replaced at 2-week intervals. This makes the administration of
GH labour-intensive and amenable only to intensive production systems
such as the dairy industry.
Studies on the effects of GH on the growth performance of cattle have
proven disappointing. The effects of GH in these animals are small and
inconsistent. Daily injections or weekly implants of GH into finishing steers
or heifers over 3-month periods demonstrated dose-dependent increases in
ADG and feed efficiency, while reducing feed intake. In steers, GH injected
daily at 16.5 to 33 µg/kg body weight/day increased ADG by about 10%
and feed efficiency by 10% to 20%, and reduced feed intake by 2% to 5%.
Higher doses of GH (100 µg/kg/day) affected only feed efficiency, while
300 µg/kg/day reduced gain, intake and efficiency. For comparison, anabolic
steroid implants containing oestradiol benzoate and trenbolone acetate
(TBA) induced an increase of about 30% in ADG and a 20% to 35% in feed
efficiency while reducing feed intake to be about 5%. Small or no effects of
GH treatment on final body weights, carcass weight, loin eye area or backfat
thickness have been reported. Effects of GH in meat ruminants are limited
by lack of adequate nutrition, especially available amino acids. Animals
must receive diets which are higher in rumenal escape protein, essential
amino acids and energy to support the increased protein accretion rates and
intake induced by GH. Even in studies in which these energy sources are
provided, the effects on ruminant growth are minimal. As a result, GH has
not been adopted in beef cattle production systems.
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Chapter 5
2.9
2.7
2.5
2.3
2.1
1.1
1.0
0.9
360
160
300
240
180
120
60
140
120
100
80
60
30
Ash accretion (g/day)
3.1
Feed/gain ratio
Liquid accretion (g/day)
3.1
Protein accretion (g/day)
Avg. daily gain (kg/day) Avg. daily feed intake (kg/day)
In contrast to the minimal effects of GH in beef cattle, the effects of GH
on the growth and body composition of swine are dramatic. Treatment of
pigs with GH induces large increases in feed efficiency (with reduced feed
intake), ADG, carcass protein and lean carcass components and reduces
body fat in a dose-dependent manner (Fig. 5.12).
Many experiments with growing pigs have shown that when GH was
injected at 100 µg/kg body weight/day, ADG increased by 10% to 20%, feed
intake was reduced by 15% and feed efficiency increased by 13% to 33%. At
the same time, lipid accretion was reduced by up to 79% and protein deposition was increased by up to 62%. It is possible that these tremendous effects
on body composition and nutrient partitioning from fat to muscle deposition
from GH treatment may be mediated by the IGFs. A study that looked at the
2.9
2.7
2.5
2.3
2.1
0
30 60
120
200
20
10
0
30 60
120
200
Somatotropin dose(µg/kg/day)
Fig. 5.12. Dose–response effects of GH in growing swine. (With permission from Campion
et al., 1989. Animal Growth Regulation, Plenum Press.)
Growth Hormone and Insulin-like Growth Factors
119
effects of IGF-I or GH treatment, alone or in combination, of pre-pubertal
barrows has been done. The results of this study are shown in Table 5.3.
In this study, which used optimal doses of GH, treatment with GH or
IGF-I did not alter feed intake. All treatments increased ADG and slaughter
weight, but the effects of GH were much greater than those of IGF-I. GH had
a large positive effect on feed efficiency, while IGF-I treatment did not affect
feed efficiency. While IGF-I stimulated a 33% increase in carcass protein, the
GH treatment induced an 88% increase in carcass protein. Only a small
increase in lean cuts was induced by IGF-I, while GH treatment resulted in
a 16% increase. IGF-I increased perirenal fat, carcass fat, kidney and pancreas weights. All vital organ weights (heart, lung, liver, kidney, spleen) were
increased by GH treatment. The combined treatment of GH and IGF-I
resulted in effects that were not different from GH given alone. This study
suggested that treatment of growing barrows with GH induced significant
positive effects on swine growth production characteristics. GH was much
more effective than IGF-I in increasing lean body mass and protein deposition, while the effects of IGF-I were minimal. In these animals, it appears that
the effects of GH were not mediated by endocrine IGF-I and were likely due
to direct effects of GH. The local production and stimulation of tissues by
autocrine/paracrine IGF-I, under GH control, may play a role in the
observed effects of GH.
GH and the development of transgenic animals
The GH gene was the first to be used to develop a transgenic animal that
overexpressed the product of a gene foreign to the natural genome of a
mammal. In 1982, Ralph Brinster’s group at the University of Pennsylvania
combined the bGH gene and the mouse metallothionein-I promoter in a bacterial plasmid (Palmiter et al., (1982)). The metallothionein promoter allowed
the induction of the GH gene in response to the metals zinc or cadmium.
This recombinant DNA was then injected into the pronuclei of mouse eggs
where it inserted itself into the mouse DNA. Roughly 600 copies of this gene
were injected into each egg. A total of 170 resulting eggs were transferred
into foster mothers and allowed to come to term. Of the 170 transferred eggs,
Table 5.3. Effects of IGF-I or GH treatment, alone or in combination, in swine (%).
Treatment (dose)
ADG
FE
Carc. prot.
Lean cuts
IGF-I (2 × 33 µg/kg/day)
+22
n.s.
+33
+5
pGH (33 µg/kg/day)
+43
+60
+88
+16
IGF-I + pGH (33+33)
Same as GH alone
Adapted from Klindt et al. (1998).
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Chapter 5
21 animals developed to term and survived parturition. Seven of the surviving animals were positive for the presence of the GH–metallothionein construct. Breeding studies showed that only one mouse was capable of
transmitting the gene in a Mendelian manner to its offspring.
Postnatal growth of the transgenic mice was proportional to the number
of copies of the GH gene present in the tissues and to the expression of GH
mRNA. There was a great deal of individual variation in the incorporation
of the GH gene and tissue expression of GH mRNA. The serum concentrations of GH in four of the seven transgenic mice were 100–800 times greater
than in control littermates and body weights were up to double those of control,
non-transgenic littermates. Gigantism was observed in the transgenic mice
and females were infertile. The animals’ livers were enlarged and many had
hepatic lesions. In addition, the animals were insulin-resistant, displaying
the characteristics of Type II diabetes.
This pioneering study demonstrated that a foreign gene could be successfully transferred into a mammal. Unfortunately, there were many problems with these transgenics. For example, the GH gene is normally
expressed only in specific cells of the anterior pituitary, and in the transgenic
animal GH is incorporated and expressed by all tissues of the animal. This
can lead to high local concentrations of GH in tissues that are not usually
exposed to this extreme of GH concentration. Expression of the gene was
largely unregulated, despite the presence of the metallothionein gene, and
massive pharmacological levels of circulating GH were observed in the
transgenic animals. The expression and function of GH in mammals is regulated by nutrients and developmental stage, while the regulation of GH
expression in the transgenic mice was uncontrolled by development or
nutrition. This study showed that the regulation of foreign gene expression
in animals needed to be regulated to avoid constant, high levels of the gene
product at the organ and systemic level. A regulatory system that controls
gene expression in a manner similar to that of the natural gene is required to
reap the benefits of transgenic animals.
A study was reported in 1990 by Richard Hanson’s group at Case
Western Reserve University (McGrane et al., (1990)) that was designed to
overcome the problems seen with the unregulated expression of GH in the
GH/MT-I transgenic mice. This group spliced bGH gene to the promoter for
the hepatic enzyme phosphoenolpyruvate carboxykinase (PEPCK). This
gene is expressed in a tissue-specific manner, and is controlled by nutrients,
hormones and the developmental stage of the animals. By combining this
promoter with the GH gene, it was hypothesized that a more controlled
expression of the transgenic gene could be attained.
PEPCK catalyses the first step in hepatic gluconeogenesis, the synthesis
of glucose from non-carbohydrate sources. PEPCK activity is localized to the
adult liver, kidney and small intestine, but some activity is also detectable in
the mammary gland, fat and skeletal muscle. The mammalian fetus receives
all of its glucose by placental transfer from maternal sources. Therefore, the
PEPCK gene is not expressed in the fetus, as there is no requirement for
endogenous glucose synthesis. During fetal development, elevated insulin
Growth Hormone and Insulin-like Growth Factors
121
and glucose levels inhibit expression of the PEPCK gene. At birth, when the
connection to maternal sources of glucose is severed and gluconeogenesis
becomes important, the PEPCK gene is turned on. This occurs when neonatal levels of insulin and glucose drop and the concentrations of glucagon and
glucocorticoid (cortisol) increase.
The PEPCK promoter region contains several response elements that
regulate the expression of the PEPCK gene (Fig. 5.13). The promoter contains
an insulin response element (IRE). Insulin inhibits PEPCK expression via the
TK cascade, which activates a transcription factor that negatively regulates
the PEPCK gene. A glucocorticoid response element (GRE) and a thyroid
hormone response element (TRE) on the PEPCK gene promoter respond to
the respective nuclear receptors, which act as transcription factors, for these
hormones. Glucagon stimulates the PEPCK gene via the cAMP cascade and
the cAMP response element-binding protein (CREBP) that binds to the CREI
sequence. Tissue specificity of the PEPCK gene expression is conferred by
the P2 site, which binds hepatic nuclear factor-1. In addition, promoter-binding sites for C/EBP and TFIID (transcription factor IID) are also present in
the PEPCK promoter region.
In transgenic mice, expression of the PEPCK/bGH gene was limited to
a specific area of the liver, the periportal hepatic cells. Its expression was also
regulated developmentally. The PEPCK promoter-driven GH gene was inactive in the fetus, but expressed in the neonatal and adult animal. The expression of GH was repressed by postprandial elevations of insulin and glucose
inhibited GH secretion in the transgenic mice. Gene expression of GH in the
PEPCK/bGH transgenic mice was induced by glucagon and cortisol.
Generation of transgenic animals with a promoter region that responds in a
relatively physiological manner to changes in hormones, nutrients and
developmental stage illustrates the complexity of the control of gene expression and provides an animal in which the gene is more closely regulated in
a tissue-, developmental and nutrient-dependent manner.
Fig. 5.13. The promoter region of the PEPCK gene. The lower scale indicates the number of
base pairs of the promoter region preceding the PEPCK gene.
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Chapter 5
References and Further Reading
Dalke, B.S., Roeder, R.A., Kasser, T.R.,
Veenhuizen, J.J., Hunt, C.W., Hinman,
D.D. and Schelling, G.T. (1992)
Dose–response effects of recombinant
bovine somatotropin implants on feedlot
performance in steers. Journal of Animal
Science 70, 2130–2137.
Eigenmann, J.E., Patterson, D.F. and Froesch,
E.R. (1984) Body size parallels insulinlike growth factor I levels but not growth
hormone secretory capacity. Acta
Endocrinology 106, 448–453.
Etherton, T.D. and Bauman, D.E. (1998)
Biology of somatotropin in growth and
lactation
of
domestic
animals.
Physiological Reviews 78, 745–761.
Green, H., Morikawa, M. and Nixon, T. (1985)
A dual effector theory of growth-hormone action. Differentiation 29, 195–198.
Hossner, K.L., McCusker, R.H. and Dodson,
M.V. (1997) Insulin-like growth factors
and their binding proteins in domestic
animals. Animal Science 64, 1–15.
Isaakson, O.G.P., Jansson, J.-O. and Gause,
I.A.M. (1982) Growth hormone stimulates longitudinal bone growth directly.
Science 216, 1237–1239.
Klindt, J., Yen, J.T., Buonomo, F.C., Roberts,
A.J. and Wise, T. (1998) Growth, body
composition and endocrine responses to
chronic administration of insulin-like
growth factor I and (or) porcine growth
hormone in pigs. Journal of Animal
Science 76, 2368–2381.
LeRoith, D., Bondy, C., Yakar, S., Liu, J.L. and
Butler, A. (2001) The somatomedin
hypothesis: 2001. Endocrine Reviews 22,
53–74.
McGrane, M.M., Yun, J.S., Moorman, A.F.M.,
Lamers, W.H., Hendrick, G.K., Arafah,
B.M., Park, E.A., Wagner, T.E.
and Hanson, R.W. (1990) Metabolic
effects of developmental, tissue-, and
cell-specific expression of a chimeric
phosphoenolpyruvate carboxykinase
(Grp)/bovine growth hormone gene in
transgenic mice. Journal of Biological
Chemistry 265, 22371–22379.
Moseley, W.M., Paulissen, J.B., Goodwin,
M.C., Alaniz, G.R. and Claflin, W.H.
(1992) Recombinant bovine somatotropin improves growth performance
in finishing beef steers. Journal of Animal
Science 70, 412–425.
Palmiter, R.D., Brinster, R.L., Hammer, R.E.,
Trumbauer, M.E., Rosenfeld, M.G.,
Birnberg, N.C. and Evans, R.M. (1982)
Dramatic growth of mice that develop
from eggs microinjected with metallothionein–growth hormone fusion genes.
Nature 300, 611–615.
Powell-Braxton, L., Hollingshead, P.,
Warburton, C., Dowd, M., Pitts-Meek,
S., Dalton, D., Gillett N. and Stewart,
T.A. (1993) IGF-I is required for normal
embryonic growth in mice. Genes and
Development 7, 2609–2617.
Salmon, W.D. and Daughaday, W.H. (1957) A
hormonally controlled serum factor
which stimulates sulfate incorporation
by cartilage in vitro. The Journal of
Laboratory and Clinical Medicine 49,
825–836.
Schlechter, N.L., Russell, S.M., Spencer, E.M.
and Nicoll, C.S. (1986) Evidence suggesting that the direct growth-promoting
effect of growth hormone on cartilage
in vivo is mediated by local production of
somatomedin. Proceedings of the Natural
Academy of Sciences USA 83, 7932–7934.
Schoenle, E., Zapf, J., Humbel, R.E. and
Froesch, E.R. (1982) Insulin-like growth
factor I stimulates growth in hypophysectomized rats. Nature 296, 252–253.
Schoenle, E., Zapf, J., Hauri, C., Steiner, T. and
Froesch, E.R. (1985) Comparison
of in vivo effects of insulin-like growth
factors I and II and of growth
hormone in hypophysectomized rats.
Acta Endocrinology 108, 167–174.
6
Calcium Homeostasis
and Regulation of Bone Growth
Bone provides the structure for muscle attachment and locomotion and
allows us to maintain our body form. Although one does not usually consider bone as a physiologically active organ, bone is an active organ system
that respires, metabolizes substrates and is in a constant flux, growing and
changing shape throughout an animal’s lifetime. In adult humans, the entire
skeleton is replaced about every 10 years. Bone is continually changing
shape, especially in the growing animal. The shapes of bones are altered by
removing bony matrix through the process called bone resorption and
replacing the resorbed bone by synthesis and deposition of new bone. The
processes of bone resorption and deposition are tightly linked and existing
bone is dissolved and resorbed by osteoclasts at the same time as new bone
matrix is deposited by osteoblasts. Bone also acts as a reservoir or storage
site for several important minerals, especially calcium and phosphate, which
are found in the circulation.
Bones contain 99% of the body’s calcium and 85% of the phosphate.
Calcium and phosphate play important roles in regulating homeostatic
processes in animals. For example, calcium is an important cofactor in the
process of blood clot formation and, along with Na+ and K+, helps to
maintain the transmembrane potential of cells. This is essential for maintenance and regulation of nerve and muscle excitability as well as the
maintenance of all cellular functions. In addition, calcium serves as a
cofactor for several enzymes and, as we have seen, is essential in the activation of many intracellular messengers for the endocrine system.
Phosphate is an important component in many biological molecules,
including nucleotides, nucleic acids, phospholipids, proteins and
enzymes. Calcium and phosphate are stored in bone as hydroxyapatite,
Ca10(PO4)6(OH)2, the insoluble form of the bone mineral matrix. After
deposition by osteoblasts and osteocytes, hydroxyapatite precipitates on
the organic collagen matrix in alkaline environments. Hydroxyapatite is
solubilized in acidic environments, in response to reduced blood Ca2+.
This chapter will first outline the effects of hormones on calcium homeostasis and then will examine the regulation of bone growth and differentiation by hormones and growth factors.
©K.L. Hossner 2005. Hormonal Regulation of Farm Animal Growth (K.L. Hossner)
123
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Chapter 6
Hormonal Regulation of Calcium Homeostasis
Regulation and maintenance of blood calcium levels involves a balance
between sources of circulating calcium and mechanisms to remove calcium
from the bloodstream. Calcium in the circulation is derived from calcium
ingested in the diet and absorbed by the small intestine, and from mobilization of calcium from the skeleton. Regulation of blood calcium levels occurs
at the organ level in the intestinal epithelium and the kidneys. In the intestinal epithelium, calcium from the diet is actively transported into the circulation. The kidneys regulate circulating calcium concentrations through the
processes of calcium excretion and reabsorption (Fig. 6.1). These processes,
essential for the maintenance of calcium homeostasis, are regulated in large
part by the endocrine system. The three major hormones which regulate the
intestinal uptake, renal excretion and bone homeostasis of calcium and phosphate are parathyroid hormone (PTH), 1,25-dihydroxy vitamin D3 (1,25(OH)2-D3) and calcitonin (CT). Both PTH and vitamin D elevate blood
calcium concentrations in response to lowered serum calcium, while CT
reduces blood calcium in response to elevated calcium. The relationships
between these hormones are shown in Fig. 6.2.
Parathyroid hormone
PTH is synthesized by the four parathyroid glands, two of which are embedded on the back of each of the paired thyroid glands. The presence of PTH is
essential for life. While other hormones are needed for normal body function, they are not essential for survival. The essential nature of PTH is
Fig. 6.1. Homeostatic regulation of blood calcium.
Calcium Homeostasis and Regulation of Bone Growth
125
Fig. 6.2. Hormonal regulation of calcium homeostasis.
demonstrated by the consequences of removing PTH. Historically, when the
thyroid glands were surgically removed, the existence of the parathyroid
glands was unknown and these were unwittingly removed along with thyroids. This resulted in a rapid decline in blood calcium, followed by tetanic
convulsions and death.
PTH is a single-chain polypeptide consisting of 84 amino acids. Its
molecular weight is ∼9600. PTH is released from the parathyroid glands in
response to reduced blood calcium concentrations. The primary targets for
PTH are the kidneys and osteoblast cells in the bone. The biological effects
of PTH are mediated by membrane-bound G-protein-linked PTH receptors
in target tissues.
Biologically, PTH has several effects on calcium homeostasis. In general,
PTH induces an elevation of blood calcium, while reducing blood phosphate. It does this through three separate mechanisms. In the kidney, PTH
has a direct effect, increasing tubular reabsorption of calcium while increasing phosphate excretion. PTH also has an indirect effect on the kidney, where
it stimulates 1,25-(OH)2-D3 synthesis. 1,25-(OH)2-D3, in turn, acts on the
small intestine to increase calcium absorption from the diet. In bone, PTH
enhances bone resorption and the release of calcium and phosphate by
effects on osteoblasts and osteoclasts. As bone resorption is the primary
function of osteoclasts, it was thought that PTH effects on bone resorption
were due to the direct effects of PTH on osteoclasts. Like many hormones
and growth factors that stimulate bone resorption by osteoclasts, the effects
of PTH are indirect, and mediated by osteoblasts. Osteoblasts, after stimulation by PTH, activate osteoclasts to undergo differentiation and initiate bone
resorption. This unique relationship between osteoblasts and osteoclasts was
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Chapter 6
Table 6.1. Demonstration of the indirect effect of PTH on bone resorption.
Cells/hormone
Surface area resorbed (µm2 ×10−3)
Osteoclasts alone
3.8
Osteoclasts + PTH
4.8
Osteoclasts + osteoblasts
2.8
Osteoclasts + osteoblasts + PTH
8.4
Source: McSheehy and Chambers (1986).
first demonstrated using an in vitro system to study bone resorption. In this
system, isolated osteoblasts and osteoclasts, alone or in combination, are
grown on slices of human femoral bone. The bone surfaces were then evaluated for the amount of bone that was degraded by measuring the surface
area of the osteoclast-created pits created by osteoclasts during the incubation period (Table 6.1).
This study showed that when osteoclasts alone were treated with PTH,
there was no increase in bone resorption over that of untreated osteoclasts.
In addition, the co-culture of osteoblasts and osteoclasts, without hormonal
stimulation, did not induce bone resorption. However, when osteoblasts and
osteoclasts were grown together and stimulated with PTH, the bone resorption area was more than doubled compared to the untreated co-cultures.
Thus, PTH does not act directly on osteoclasts to increase bone resorption.
The induction of osteoclastic bone resorption by PTH requires the presence
of osteoblasts. PTH-stimulated osteoblasts, in turn, signal osteoclasts to
increase bone resorption. It is now known that this interaction between PTH,
osteoblasts and osteoclasts is mediated by a unique cell-to-cell interaction
system, the RANK/RANKL system, which was discussed in Chapter 4.
1,25-Dihydroxy vitamin D3
1,25-Dihydroxy vitamin D3, commonly known as vitamin D, is a steroid that
is synthesized within the body, circulates in the bloodstream and acts via
specific receptors in target cells. It is an essential regulatory factor in maintaining the homeostasis of blood and bone calcium. Vitamin D does not fulfil the classical definition of a vitamin, that is, a substance essential for
normal metabolism and growth that is provided by dietary sources. Vitamin
D is synthesized in the body and external sources are not normally required
for adequate metabolism. However, as the precursor vitamin D was first
identified as an essential nutrient, it is still classified as a fat-soluble vitamin.
Vitamin D might be better described as a prohormone, as it is a precursor to
the biologically active hormone, 1,25-(OH)2 vitamin D3.
As shown in Figs. 6.3 and 6.4, the synthesis of active 1,25-(OH)2 vitamin
D3 is accomplished by the cooperation of multiple organs. The steroid pre-
Calcium Homeostasis and Regulation of Bone Growth
127
Fig. 6.3. 1,25-(OH)2-Vitamin D3 is synthesized by multiple organs.
cursor, 7-dehydrocholesterol, is a cholesterol derivative that is converted in
the epidermis into the prohormone vitamin D3 (also called cholecalciferol), a
reaction that is catalysed by the ultraviolet portion of sunlight. In the bloodstream cholecalciferol is bound to a protein called vitamin D-binding protein
(VDBP). In the liver, cholecalciferol is hydroxylated to form 25-hydroxy vitamin D3. This, in turn, is transported by VDBP in the circulation to the kidney,
where it is further hydroxylated in the mitochondria by the enzyme
1α-hydroxylase into the active 1,25-(OH)2-D3. Another enzyme in the kidney, 24-hydroxylase, can convert the 25-hydroxy vitamin D3 precursor into
an inactive metabolite, 24,25-(OH)2-D3.
Biological actions of 1,25-dihydroxy vitamin D3
1,25-Dihydroxy vitamin D3, or vitamin D, acts via a nuclear steroid receptor in
its target tissues. Binding of vitamin D activates the vitamin D receptor (VDR)
and the receptor forms dimers with other VDR molecules. This activated receptor complex then interacts with specific enhancer and promoter sequences of
specific genes in the nucleus to activate or repress their expression.
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Chapter 6
Fig. 6.4. Chemical structure and synthesis of biologically active 1,25-(OH)2 vitamin D3.
The main target organs affected by vitamin D are the small intestine, the
kidney and bone. The primary effect of vitamin D is in the intestine, where it
stimulates the uptake of dietary calcium by the intestinal epithelium. The
trophic effect of vitamin D on bone mineralization may actually be a secondary
or indirect effect of vitamin D. Thus, the effects on bone growth and mineral-
Calcium Homeostasis and Regulation of Bone Growth
129
ization may be due simply to the provision of adequate dietary calcium for
bone formation. For example, in knockout mice lacking the VDR or in vitamin
D-deficient individuals, the effects of vitamin D deficiency on bone can be overcome simply by providing supplementary calcium and phosphate in the diet.
In the intestine, vitamin D enhances calcium uptake from the diet by
increasing the synthesis of an intracellular calcium transport protein called calbindin. Calbindin ferries calcium from the intestinal luminal side of the cell to
the basal portion of the cell. Calcium transport out of the intestinal epithelial
cell occurs against a concentration gradient, and secretion from the cell requires
the active transport of calcium by a calcium pump. Vitamin D also induces the
synthesis of the calcium pump mRNA and protein. Hence, vitamin D induces
the uptake and delivery of dietary calcium into the bloodstream.
Vitamin D is required for bone mineralization. Animals deficient in vitamin D are characterized by the development of poorly mineralized bones. In
children, this results in a syndrome called rickets, while in adults vitamin D
deficiency causes a similar pathological state called osteomalacia. Both result
in mineral-deficient, soft, flexible bones that are incapable of adequately supporting the body and deform under the normal stress of body support. While
the effects of vitamin D on mineral homeostasis in vivo are well known and
are centred on the stimulation of bone mineralization, vitamin D treatment of
bone cells in vitro stimulates the formation of osteoclasts. This effect of vitamin D on osteoclast formation is indirect, mediated by the VDR in osteoblasts
and the subsequent expression of RANKL in osteoblasts. Activation of the
RANK/RANKL system in osteoblasts and osteoclasts stimulates the differentiation and activation of osteoclasts and results in bone resorption. As bone
mineralization and remodelling is coupled to bone resorption, activation of
osteoclasts by vitamin D is likely a complementary portion of the bone
remodelling system, as bone synthesis is coupled to bone resorption. Vitamin
D also stimulates the production of the non-collagenous matrix proteins,
osteocalcin and osteonectin, in osteoblast cultures in vitro. Thus, in addition
to its role in calcium and phosphate homeostasis, vitamin D has effects on the
skeleton that are related to bone remodelling and bone turnover.
The production of vitamin D is stimulated by calcium and phosphate
deficiencies. Reduction of phosphate is detected in the kidney, which
increases vitamin D synthesis, while the effects of reduced calcium are mediated by an increase in PTH secretion, which stimulates vitamin D synthesis
and secretion by the kidney. Elevated levels of vitamin D enhance the PTHstimulated reabsorption of calcium by the distal tubules by increasing the
synthesis of the PTH receptor in the distal tubule. Elevated calcium levels, as
well as elevated vitamin D itself, directly inhibit the vitamin D 1α-hydroxylase enzyme in the kidney and reduce the synthesis of 1,25-(OH)2-D3.
Calcitonin
CT is the third major hormonal player in the regulation of bone and blood
calcium. The primary source of CT is the C-cells (parafollicular cells) of the
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Chapter 6
thyroid gland. The major cell types of the thyroid gland are the follicular
cells, which are responsible for thyroid hormone secretion. The parafollicular cells comprise a minor proportion of thyroid gland cells and are dispersed throughout the thyroid gland, but occur mainly in the central region
of the gland. In addition, CT has been found in several organs in the body,
including the thymus, small intestine, urinary bladder, lung and liver in
humans. CT is a small polypeptide, consisting of 32 amino acids, with a
molecular weight of 3410 Da. CT is a product of the CT gene family, which
consists of five genes: CALC I, II, III, IV and V. CALC-I is the source of
human CT. These genes also produce other family members including CT
gene-related peptides I and II, adrenomedullin, amylin and other peptides
present in the circulation. Members of this family are characterized by a single disulphide bond that links the amino terminal cysteine and a cysteine at
position 7, forming a ring structure containing seven amino acids. The carboxy terminal end of this family of peptides is characterized by the presence
of an amide cap.
The plasma membrane receptor for CT was cloned in 1991 from porcine
kidney cells. The receptor belongs to the G-protein-linked receptor family. It
is a seven transmembrane-domain receptor that consists of 482 amino acids.
Activation of the receptor by CT binding induces several different intracellular transduction pathways, including induction of adenylate cyclase and
cAMP production, and activation of PKA as well as the phosphoinositidedependent pathway involving PLC and intracellular Ca2+ mobilization.
Activation of a specific pathway depends not only on target cell type, but
also may vary according to the stage of the cell cycle of the target cell. CT
receptors are widespread throughout the body, occurring in the CNS and
reproductive tissues, where their function is largely unknown. The distribution of CT receptors and CT-producing cells in the body suggests a wider
role of CT in general metabolism. Of interest to the metabolism and homeostasis of calcium, CT receptors are present in the major target organs, the
kidneys and the osteoclasts.
CT was initially isolated in the 1960s as a hypocalcaemic factor, on the
basis of its ability to reduce serum calcium concentrations. The precise role
of CT in the normal physiological regulation of calcium remains uncertain.
Studies of the function of CT are flawed by the use of CT derived from
unique animal species, including the salmon and eel, the use of rodents, the
widespread synthesis of CT in the body and the use of pharmacological
doses to elicit responses. CT is released into the bloodstream in response to
Ca2+ infusion. When animals are treated with CT, there is an acute reduction
of serum levels of calcium, but the magnitude of this response varies with
animal and CT species, dose and injection route. CT reduces blood calcium
levels by increasing the excretion of calcium by the kidneys and by the inhibition of bone resorption.
CT inhibits bone resorption by direct effects on osteoclasts, initially
inhibiting the motility of osteoclasts, followed by the retraction of osteoclast
pseudopods that contact the bone surface. Then it is followed by a CTinduced alteration of the characteristic morphology of the osteoclasts and
Calcium Homeostasis and Regulation of Bone Growth
131
the osteoclasts form small, rounded and non-motile cells. These morphological changes are accompanied by loss of the typical biochemical profile of the
osteoclasts. The carbonic anhydrase activity and the acid phosphatase activity, characteristically elevated in active osteoclasts, are reduced by very small
concentrations (picomolar) of CT. Chronic treatment with CT reduces the
number of osteoclasts in bone as well as the levels of urinary hydroxyproline, an amino acid marker of bone resorption. Because of the effects of CT
on reducing osteoclast activity and function, CT is used to treat bone loss
and rapid bone turnover diseases. It is also used to correct hypercalcaemic
disorders that result from mobilization of calcium from the skeletal system.
Regulation of Bone Growth and Development
The hedgehogs and early skeletal differentiation
Cell-to-cell signalling is a critical component of tissue and organ development in the early embryo. Only five signal transduction pathways control
the early development of tissues in most animals. These cellular signals consist of wingless (Wnt), TGF-β, notch, receptor tyrosine kinase (RTK) and the
hedgehogs. The hedgehogs act as paracrine agents secreted by embryonic
cells that induce differentiation of specific adjacent tissues during embryonic
development. These molecules are called morphogens due to their distinct
effect on inducing morphological changes. The hedgehogs, notch and wingless were first discovered in the fruit fly, Drosophila melanogaster, and were
named after the mutant fruit fly phenotypes.
The hedgehogs determine the fate of undifferentiated precursor cells,
providing inductive cues to these cells and committing them into specific
differentiation pathways. Embryos with the hedgehog mutation are covered
with pointed denticles, resembling a hedgehog. At present, there are three
known hedgehog genes in vertebrates: sonic hedgehog (SHH), Indian
hedgehog (IHH) and desert hedgehog (DHH). Two of the genes are named
after species of hedgehog (Indian and desert) and sonic hedgehog is named
after a video game. The hedgehogs act on target cells through specific
plasma membrane receptors with 12 transmembrane domains called
patched (Ptc). Smoothened (Smo) is a seven transmembrane co-receptor that
does not bind hedgehogs, but inhibits the constitutive activation of the Ptc
signal. Binding of the hedgehog ligand to Ptc removes the constitutive inhibition of Smo and allows induction of intracellular signals and the subsequent activation of specific genes. The hedgehogs have been implicated in
the differentiation and development of major portions of the body, including
the brain, spinal cord, eyes, limbs and craniofacial regions as well as the
skeleton. In the skeleton, SHH is expressed in the embryonic limb bud where
it induces mesenchymal determination and limb formation. SHH also
induces early chondrocyte differentiation and osteocyte maturation. IHH
acts as a mitogen in the skeletal system, stimulating chondrocyte proliferation while slowing differentiation.
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Chapter 6
Regulation of bone growth and differentiation by hormones
Unlike the hormones discussed above, PTH, CT and vitamin D, which regulate mineral homeostasis in bone and blood, the hormones described in this
section are involved with bone cell replication, differentiation, maturation
and maintenance of the differentiated state. In this section, the effects of the
GH/IGF system, glucocorticoids, sex steroids and thyroid hormones on
bone growth and development are discussed.
The GH/IGF-I system is a primary regulatory system in bone, and is
essential for the development and maintenance of the differentiated function
of bone tissue. Many of the effects of the IGF/GH system are discussed in
Chapter 5. Several studies have been conducted to distinguish between the
effects of GH and IGF-I on bone. The complexity of the GH/IGF system
makes it difficult to separate the action of these factors. GH and IGF-I act as
hormones and circulating concentrations of these factors have effects on distant target tissues. In addition, the IGFs are produced locally in most, or all
of the tissues in the body, where they act as growth factors without the intervention of the circulatory system. Before the mid-1980s, there was a great
deal of controversy about the role of endocrine GH and IGF-I in stimulating
long bone growth. This controversy was settled when Isaksson showed that
the effects of GH on long bone growth were mediated by local bone production of IGF-I. It is now believed that GH stimulates bone precursor mesenchymal cells to differentiate and express IGF-I and the IGF-I receptor.
IGF-I then acts in an autocrine manner to enhance clonal mitotic growth of
these cells and to maintain their differentiated function. This is known as the
dual effector hypothesis of GH and IGF action, as described in Chapter 5.
Recent studies have shown that IGF-I and IGF-II are found in abundance
in bone and that the IGFs play critical roles in the regulation of bone growth
and development. Although the IGFs are also present in the circulation at
high concentrations, the local secretion and activity of IGFs in the bone are
believed to be the primary mechanism by which IGFs regulate bone growth.
Both IGF-I and IGF-II are present in the bone, but the activities of one form
vs the other have not been adequately examined. The majority of studies
have used cells derived from laboratory rodents or humans and the effects
of IGF-I and IGF-II vary according to species, cell type and the stage of differentiation of the cells studied. Both IGF-I and IGF-II are synthesized and
secreted by osteoblasts. They are stored in the bony ECM and their effects
are modulated by the presence of IGF-binding proteins in the matrix. The
actions of both forms are believed to be mediated by the Type I IGF receptor,
which is present in osteoblasts and osteoclasts.
Both IGF-I and IGF-II can induce the formation of bone in vivo and
in vitro. The original studies that identified the IGFs as growth factors used
a cartilage system to show that factors induced by GH were responsible for
cartilage and bone growth. The IGFs are generally thought of as mitogenic
factors, but in bone they also play important roles in the recruitment of precursor cells to the osteoblast and osteoclast cell lineage and function in the
maintenance of the differentiated state of bone. The IGFs stimulate the pro-
Calcium Homeostasis and Regulation of Bone Growth
133
liferation of pluripotent bone marrow stromal cells, thus providing an
increased population of precursor cells available for differentiation into
osteoblasts. IGFs do not induce differentiation of these cells, as indicated by
the lack of induction of a specific transcription factor, Cbfa1, that is essential
for commitment of precursor cells to the osteoblast lineage. Commitment
and differentiation of these cells is regulated by other growth factors and
morphogens. After differentiation into osteoblasts, the IGFs induce the synthesis of typical proteins characteristic of the differentiated state, such as
Type I collagen and other matrix proteins. The IGFs also reduce collagenase
expression by osteoblasts. Thus, the effects of IGFs on bone cells depend
upon the state of differentiation of these cells. The IGFs act as mitogens for
the undifferentiated precursor cells and maintain the differentiated function
of mature osteoblasts.
The IGFs also have direct and indirect effects on osteoclasts, inducing
recruitment, formation and activation of these bone-resorbing cells. The IGFs
stimulate migration of precursor haematopoietic cells, the formation of the
multinucleated osteoclasts and the secretion of tartrate-resistant acid phosphatase, a biochemical marker of activated osteoclasts. In mature osteoclasts,
IGF-I, but not IGF-II, induces bone resorption. This effect is indirect, as
osteoblasts must be present for induction of bone resorption, and may be
mediated by the intercellular osteoblast–osteoclast RANK/RANKL system
described in Chapter 4.
The expression of IGFs by osteoblasts varies with developmental stage
and the differentiation state of these cells. As osteoblasts differentiate, the
secretion of IGF-I is reduced while IGF-II secretion is increased. This has led
to the suggestion that IGF-II may be the more important IGF in bone cells,
but this has not been conclusively demonstrated. Circulating hormones and
local growth factors also regulate the secretion of the IGFs from osteoblasts.
The effects of PTH on bone growth and mineralization are mediated in part
by IGF-I, as PTH treatment of osteoblasts increases IGF-I expression in vitro
and in vivo. Glucocorticoids, which induce differentiation of bone marrow
stromal cells into osteoblasts, suppress the synthesis of IGF-I, while increasing IGF-II expression. Other growth factors in the bone matrix also regulate
the expression of the IGFs. For example bone morphogenic proteins (BMP),
which induce proliferation and differentiation of osteogenic cells, induce
IGF-I and IGF-II expression in osteoblasts. Potent mitogens such as fibroblast
growth factor (FGF) and PDGF inhibit the expression of IGFs in osteoblasts.
The effects of glucocorticoids on bone are largely dependent upon dose.
Large, pharmacological amounts of glucocorticoids are catabolic to bone
formation and induce bone loss. This is seen during treatment of individuals with glucocorticoids to reduce inflammation or to suppress the immune
system. Cushing’s disease, characterized by the oversecretion of glucocorticoids from the adrenal gland, also induces bone loss in affected individuals.
These effects may be due to indirect actions of the glucocorticoids, which
reduce GH, oestrogen and testosterone secretion in the intact animal.
Pharmacological doses of glucocorticoids also reduce intestinal absorption
of calcium and increase calcium and phosphate excretion in the urine.
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Chapter 6
Glucocorticoids have direct effects on the kidneys, and also act indirectly, by
increasing PTH and vitamin D concentrations that augment calcium excretion.
Glucocorticoids have direct effects on bone. Osteoblasts, as well as most
cells in the body, have glucocorticoid receptors. Pharmacological doses (hundreds of nanomolar to micromolar range) of glucocorticoids inhibit preosteoblast and osteoblast DNA, RNA and protein synthesis and reduce bone
formation. Proliferation and the differentiated functions of mature
osteoblasts, such as collagen secretion, are inhibited by high doses of glucocorticoids. At physiological levels, in the low nanomolar range, glucocorticoids are anabolic and stimulate the differentiation of osteoblast progenitor
cells and enhance bone formation. At levels comparable to those seen in the
normal animal, glucocorticoids increase the differentiation of bone marrow
stromal cells into the osteoblast cell lineage. At the same time, glucocorticoids inhibit the proliferation of these cells, reducing the available pool of
progenitor cells and thus reducing bone formation. In differentiated
osteoblasts, physiological levels of glucocorticoids maintain the differentiated state, inducing Type I collagen production, alkaline phosphatase synthesis, osteoid production and bone mineralization. Glucocorticoids also
have effects on bone resorption, via the osteoclasts. Like many hormones’
effects on osteoclasts, the glucocorticoids act upon osteoblasts, inducing the
RANKL protein and decreasing the expression of the soluble decoy receptor,
OPG, in these cells. As we have seen, this results in a stimulation of osteoclast formation and, as a result, increased bone resorption.
Many of the effects of glucocorticoids in bone, as well as other tissues,
are permissive. That is, the presence of glucocorticoids is required to maintain normal cell processes, including maintenance of cell receptors for other
hormones or the provision of adequate nutrients, such as glucose, for basal
cell metabolism, growth and differentiation. For example, glucocorticoids
induce the expression of receptors for PTH and IGF-I in osteoblasts, which
allows these hormones to exert their stimulatory effects on bone. The
inhibitory effects of glucocorticoids on bone formation are, in part, mediated
by IGF-I, which is suppressed by glucocorticoid treatment. As the inhibitory
effects of glucocorticoids persist in animals lacking IGF-I (IGF-I knockout
mice), at least a portion of the effects of glucocorticoids on bone formation is
due to direct actions of the glucocorticoids in bone.
As might be expected, sex steroids have definite effects on bone growth.
The male hormone, testosterone, stimulates mitosis of cells in the growth
plate and high testosterone levels at puberty induce closure of the growth
plate. Pre-pubertal castration of males increases long bone growth due to the
combined effects of testosterone deficiency and the enhanced effects of
oestrogen. On the other hand, high doses of oestrogen reduce linear bone
growth. In bone, oestradiol-17β stimulates OPG production of the
RANK/RANKL system of osteoblasts and osteoclasts. This inhibits
RANK/RANKL interactions and inhibits bone resorption by osteoclasts.
When oestrogen is absent, as in ovariectomized animals or postmenopausal
women, the RANK/RANKL system is active and bone resorption is
increased. This results in a reduction in bone mass and bone mineralization,
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a condition known as osteoporosis. As with many hormones, oestrogen may
also have indirect effects on bone metabolism. Treatment with oestrogen
causes an increase in the circulating levels of the calcium regulatory hormones 1,25-(OH)2 vitamin D3, CT and PTH that may mediate or potentiate
some of the effects of oestrogen on bone growth and mineral deposition. In
non-ruminants, systemic IGF-I concentrations are reduced by oestrogen
treatment, while in ruminants, there is evidence that oestrogen increases circulating concentrations of GH and IGF-I.
Thyroid hormones have direct and indirect effects on most metabolic
processes in the body. They are needed to maintain metabolic rate and to
modulate oxygen consumption and mitochondrial dynamics in all cells of
the body. As with the glucocorticoids, many of the effects of thyroid hormones are likely to be primarily permissive in nature, providing a hormonal
environment that allows the cell to metabolize energy sources and respond
optimally to other hormonal stimuli. Thus, the role of thyroid hormones is
largely synergistic with other hormones and growth factors that play direct
roles in stimulating bone growth. In addition, thyroid hormones have distinct effects on bone growth. A lack of thyroid hormones during embryonic
development leads to poor bone growth, while excess thyroid hormones
cause an increase in bone resorption.
Growth factors and bone
In addition to the effects of circulating hormones, bone growth and metabolism are regulated by several polypeptide growth factors present in bone.
They are synthesized and secreted by osteoblasts and other cells in bone and
act in a local autocrine or paracrine manner in bone and cartilage. Upon
secretion, many growth factors are bound to the ECM, which contains a variety of growth factors, including several known osteoblast mitogens and differentiation-inducing factors. The growth factors in the ECM provide a
reservoir of regulatory factors, which act as autocrine and paracrine agents
to direct the growth and remodelling of bone. Growth factors are not
restricted to bone but are widely distributed throughout the body and are
present in most, if not all, tissues. In addition, the effects of growth factors
on bone growth are not tissue-specific. That is, specific growth factors are
mitogens not only in bone, but also in all tissues in which they are present.
In general, the synthesis and secretion of growth factors are not affected by
circulating hormones. Examples of these local tissue growth factors include:
FGF, PDGF, TGF, BMP as well as IGF-I and IGF-II. We will consider each of
these growth factors individually in some detail.
FGF
The FGFs are also called heparin-binding growth factors and were first isolated based on their binding to heparin using immobilized heparin affinity
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columns. The first FGFs isolated were called acidic FGF and basic FGF (aFGF
and bFGF). These forms correspond to what are now called FGF-1 and FGF2, respectively. Since the initial isolation of FGF, it has been shown that these
growth factors are part of a superfamily of polypeptides consisting of at least
23 major isoforms. These growth factors do more than simply stimulate
fibroblast mitosis. The FGFs play significant roles in embryonic development and differentiation, where they induce cell migration via chemotaxis,
and stimulate differentiation of the skeleton, skeletal muscle, neural tissue,
blood and blood vessels. The FGF superfamily of growth factors stimulates
the proliferation of a wide range of mesodermally derived and epithelial
cells, including skin keratinocytes, endothelial cells, myoblasts, osteoblasts
and chondrocytes.
The molecular weights of the FGF superfamily members range from 17
to 38 kDa, consisting of 160 to 288 amino acids. In general, the genes for the
FGF superfamily consist of three exons. Many of the FGF family members
are secreted into the extracellular space, but FGFs do not appear in the circulation. The FGFs generally exist as monomers, although dimeric forms of
some FGFs are believed to be secreted. Some FGF family members are glycosylated and many are bound to heparin and heparin sulphate in the ECM.
The ECM-bound FGF provides a local reservoir of mitogen that acts on adjacent or source cells via autocrine or paracrine mechanisms. FGFs act through
four known cell surface-bound TK receptors.
FGFs are potent mitogens and are active in picogram levels. They stimulate mitosis of a wide range of cells, such as stem cells, bone and cartilage.
In skeletal tissue, FGFs play an important role in recruitment, proliferation
and differentiation of cartilage and bone cells. During bone development the
FGFs are involved in the commitment of undifferentiated precursor cells
into the osteogenic pathway, stimulate the proliferation of preosteoblasts
and induce the differentiation of preosteoblasts into osteoblasts and finally,
osteocytes. FGF-3, -4, and -5 are expressed early in embryonic development
and in conjunction with TGF-β and BMP are responsible for early induction
of mesoderm. FGF-8 and -10 are expressed in the early embryonic limbs and
limb bud primordia and are involved in early limb development. FGF-2 and
-4 are also involved in limb bud development. FGF-1 and -2 are expressed
later in undifferentiated mesenchymal cells and in osteoblasts and chrondroblasts. The cartilage cells of the resting and proliferating zones of the
epiphyseal plate express FGF-2.
Both FGF-1 and -2 are important in bone formation and in vitro studies
show that these FGFs control osteoblast proliferation and differentiation, acting at early and late stages of osteoblast differentiation. FGF-2 is more potent
as a mitogen for osteoblasts than FGF-1. The effects of the FGFs depend
upon the stage of development and the duration of exposure to FGF. While
FGF-1 and -2 act as mitogens in early osteoblast development, later on FGF2 inhibits functions associated with the differentiated state of bone, such as
the inhibition of the synthesis and secretion of Type I collagen, osteocalcin
and alkaline phosphatase activity. In contrast, FGF-2 stimulates the expression of the matrix protein, osteopontin.
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The FGFs play crucial roles in postnatal bone growth and remodelling
in the intact animal. Treatment of growing postnatal laboratory animals
with FGF-2 stimulates bone formation and increased mineral density. The
loss of bone formation and volume that results from lack of oestrogen in
ovariectomized animals is restored by treatment with FGF-1 and FGF-2, suggesting that the FGFs may mediate the effects of oestrogens on bone. This
effect of FGF is believed to be due to recruitment and commitment of undifferentiated mesenchymal stem cells into the bone cell differentiation pathway. An important phase of bone growth is the requisite bone resorption that
occurs concurrently with bone deposition. Both FGF-1 and FGF-2 increase
bone resorption in vitro, acting via direct and indirect pathways. The FGFs
induce collagenase synthesis and secretion in bone cells. FGF-2 also induces
expression of the tissue inhibitors of metalloproteinases (TIMPs), which
inhibit collagenase activity. Thus, the FGFs have specific distinct anabolic
effects on bone formation in postnatal animals.
While many FGF effects are mediated directly, by interaction with their
cell surface receptors, some of the effects of FGFs are indirect and are likely
mediated by other locally produced bone matrix growth factors. Both FGF-1
and -2 induce TGF-β production in bone cells, in vivo and in vitro. TGF-β, in
turn, induces the expression of FGF-2 in osteoblasts, amplifying the effects
of both growth factors. FGF-2 reduces the expression of IGF-I, IGF-II and the
IGF-binding proteins while increasing expression of hepatocyte growth factor (HGF) in osteoblasts.
The effects of FGF-2 on osteoblast differentiation occur in concert with
other growth factors. For example, the effects of BMP-2 and FGF-2 are synergistic, and pre-treatment of osteoblasts with FGF-2 enhances the cell
response to BMP-2, suggesting that FGF-2 may increase BMP receptor numbers or BMP receptor sensitivity. The expression of FGF-2 in osteoblasts is, in
turn, regulated by a variety of hormones and growth factors. PTH,
prostaglandins and TGF-β are all positive inducers of FGF-2 expression in
osteoblasts. In addition, FGF-2 is autoinductive, inducing an increased
expression of its own gene in osteoblasts.
PDGF
PDGF was originally isolated from human blood platelets and was characterized by its ability to induce mitosis in a variety of cell lines, including
fibroblasts and vascular smooth muscle cells. It is now known that PDGF
is present in many different organs of the body and acts as a potent mitogen and as a chemotactic agent during development and tissue repair.
PDGF has a molecular weight of 28–35 kDa and consists of a disulphidelinked dimer of two dissimilar chains. The active form of PDGF is derived
from two separate genes that code for an A chain and a B chain. The
monomers can be combined in all combinations to produce three active
PDGF dimer isoforms: AA, AB and BB. PDGF-BB is the most potent isomer
in bone.
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The receptors for PDGF are present as monomers in the plasma membrane. Upon activation by PDGF binding the receptors form homo- and heterodimers consisting of α and β subunits: αα, αβ and ββ. The PDGF
receptors have intrinsic intracellular TK activity and, after autophosphorylation, activate PKC, PKA or induce changes in intracellular calcium.
Intracellular messenger pathways are specific to cell type. The αPDGF receptor binds all PDGF isoforms with equal affinity and the βPDGF receptor
binds PDGF-BB with about tenfold higher affinity than PDGF-AB. It does
not bind PDGF-AA. Both PDGFα and PDGFβ receptor concentrations are
upregulated by PDGF-BB.
PDGF is essential for normal embryonic development and the PDGFs
and their receptors are present throughout embryonic development. In the
early embryo, both A and B genes are expressed in epithelial tissues overlying mesenchymal tissue that expresses the α and β PDGF receptor genes.
This provides a local paracrine system that induces mitosis and migration of
mesenchymal cells. Later during embryonic development, the PDGF-A gene
is expressed by the surface ectoderm overlying limb buds. Both α and β
receptors are present in the limb bud mesenchyme and the perichondrium
of developing long bones. In the human embryo, PDGF and its receptors are
widespread, found in the placenta, blood vessels, smooth muscle and mesenchyme and throughout the CNS.
The PDGFs are not required to induce the bone phenotype per se, but
likely to play a permissive role in the embryonic development of bone.
Mutations in the PDGF-B gene or the βPDGF receptor have no specific
effects on fetal skeletal system development. Deletion of the PDGF-B gene
or the β receptor gene results in embryonic lethality. Embryos die during
the latter part of gestation (days 17–19 in mice) due to extensive vascular
haemorrhage and lack of respiratory function. More specifically, embryonic
death is due to failure of the diaphragm and kidneys to develop and to the
disorganized development of the capillary system. The βPDGF receptor
plays an important role in the development of all muscle cells including
vascular, intestinal and cardiac smooth muscle and skeletal muscle.
Disruption of the PDGF-A gene results in the death of about 50% of
embryos before embryonic day 10 in the mouse. Survivors live for a few
weeks postpartum and are severely growth-retarded. These studies demonstrate the importance of PDGF-A and -B during normal embryonic
development.
In the adult, PDGF is widely expressed in tissues derived from mesoderm, including a variety of blood cells, endothelial smooth muscle,
osteoblasts, the vascular system, neurones, fibroblasts and the kidneys.
Reproductive system tissues such as the mammary gland, uterus and placenta also produce PDGF. Although produced locally in many tissues, PDGF
acts primarily as a systemic growth factor. The blood platelets contain large
quantities of PDGF and are the primary source of circulating PDGF. PDGF is
released into the bloodstream by the platelets in response to mechanical
injury. In the circulation, PDGF induces responses to tissue injury including
inflammation, wound healing and angiogenesis.
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139
In the skeletal system, PDGF is involved in the regulation of osteogenesis and bone repair. As with many cells, PDGF is a potent mitogen for many
cell types in bone. Osteoblasts not only produce PDGF, but also they respond
to it in an autocrine manner. The mitogenic effects of PDGF are primarily on
osteoblast precursor cells such as bone marrow stromal cells, fibroblasts and
preosteoblasts. Mature osteoblasts are less responsive to the mitogenic
effects of PDGF. Treatment with PDGF results in an increase in the populations of progenitor cells which provides a pool of precursor cells that can differentiate into osteoblasts, a process that is not regulated by PDGF. The
mitogenic effects on bone precursor cells and the resulting increased progenitor cell population suggest that the primary role of PDGF in the skeletal
system is in the regulation of bone repair and osteogenesis.
PDGF has pronounced effects on bone remodelling and especially bone
resorption, acting on both osteoclasts and osteoblasts. PDGF treatment
increases the number and bone-resorbing activity of osteoclasts. Exposure of
osteoblasts in vitro to PDGF inhibits the differentiated functions of these
cells. This is reflected by the reduced alkaline phosphatase activity and the
reduced secretion of Type I collagen. In addition, PDGF reduces osteoblast
expression of specific bone ECM components such as osteocalcin and
osteonectin. As osteonectin binds and inactivates PDGF-B in the ECM,
reduction in osteonectin concentrations by PDGF may enhance the biological activity of PDGF in bone. In addition, PDGF increases the osteoblast
secretion of collagenase and gelatinase, enzymes that are involved in the
degradation of the organic matrix, or osteoid, of bone. PDGF induces the
expression of collagenases 1 and 3 in fibroblasts and osteoblasts, respectively, and reduces collagen matrix secretion in rat calvariae. Thus, PDGF is
closely associated with organic matrix degradation, a process essential in
bone resorption, remodelling and turnover.
The effects of PDGF on bone may be modulated by other growth factors.
Hormones that are active in bone such as PTH, 1,25-(OH)2-D3, cortisol and
IGF-I have no effect on the activity or binding of PDGF to osteoblasts. In contrast, growth factors such as the interleukins, TNF-α and TGF-β regulate
PDGF binding and activity in bone. Interleukins, cytokines that are primarily involved in the regulation of the growth and differentiation of blood
cells, enhance osteoblast sensitivity to PDGF by increasing expression of the
αPDGF receptor, enhancing the mitogenic effect of PDGF-AA. TNF-α also
increases PDGF binding to the αPDGF receptors. TGF-β1, on the other hand,
reduces binding of PDGF-AA and its mitogenic effects in rat and human
osteoblasts.
Effects of PDGFs on bone are also indirect, mediated by other growth
factors present in the skeleton. While the IGFs are present in large concentrations in the bone, their effects largely oppose those of PDGFs. For example, the IGFs induce Type I collagen synthesis and secretion while reducing
collagenase expression, both functions related to the maintenance of the differentiated state of osteoblasts. Treatment of osteoblasts with PDGF reduces
IGF-I and IGF-II mRNA in these cells. In osteoblasts, PDGF-BB also reduces
the expression of IGFBP-5, a binding protein that potentiates the anabolic
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actions of IGF in bone. These effects on the reduction of IGF activity in
osteoblasts enhance the effects of PDGF on collagen degradation and organic
matrix turnover.
In the intact animal, treatment with PDGF-BB prevents bone loss and
maintains spinal bone density in oestrogen-deficient ovariectomized rats.
This occurs due to an increase in osteoblast numbers with a resultant
increase in bone formation without effects on osteoclast cell numbers. As
emphasized above, osteoblast cell numbers are increased due to the effects
of PDGF on the replication of preosteoblasts and other stem cells. In the
adult animal, PDGF-AA gene expression is increased locally in many cell
types in the region of bone fractures. PDGF-BB expression is increased primarily in the osteoblasts in the area of the fracture. As PDGF is also released
in large quantities by aggregating platelets in response to bone fracture and
the accompanying tissue damage, systemic as well as locally produced
PDGF is believed to contribute to bone fracture (and wound) healing.
Transforming growth factor-β
The TGFs were originally isolated based on their effects on cell transformation. When normal cells are grown in vitro, their growth is limited by contact
inhibition. This means that when these cells contact another cell, they stop
growing. In contrast, transformed cells, such as those that have assumed a
cancerous phenotype, continue to grow when in contact with other cells.
These cells will eventually overgrow one another, forming thick, flattened
stacks of cells, similar to the uncontrolled neoplastic growth of cancer cells
seen in vivo. In addition, normal cells cannot grow when suspended in agar.
The TGFs were initially characterized by their ability to stimulate DNA synthesis, induce loss of contact inhibition and anchorage-independent cell
colony formation of cells grown in agar. Thus, the TGFs induce a state similar to that of a transformed, tumourigenic cell. The TGFs are involved in the
development of specific diseases involved with overproduction of ECM,
autoimmune diseases and carcinogenesis. The focus of this section is on the
effects of the TGFs on normal tissue development and differentiation. The
TGFs are both mitogens and inducers of differentiation in normal cells, acting via autocrine, paracrine and endocrine modes.
There are two major divisions of the TGF family: TGF-α and TGF-β.
TGF-α was first isolated from the transformed 3T3 fibroblast cell line. TGFα is a 5600 Da polypeptide that shares a 40% amino acid sequence identity
with EGF. TGF-α is considered to be an EGF-like molecule. TGF-α binds to
the EGF receptor and has mitogenic activity, similar to EGF. The focus of this
section will be on TGF-β.
The TGF-β superfamily consists of over 50 different molecules derived
from a common ancestral gene. In addition to TGF-β, this family includes
bone morphogenetic proteins, the activins, inhibins and Mullerian-inhibiting substance. The TGF-β molecules are dimers that consist of two polypeptide chains covalently linked with disulphide bonds. Three molecular
Calcium Homeostasis and Regulation of Bone Growth
141
isoforms, TGF-β1, -2 and -3, are produced by three separate genes. TGF-β1 is
the most abundant of the isoforms. Although heterodimers of TGF-β exist,
the isoforms are usually homodimers. TGF-β is secreted by cells as a large,
inactive precursor molecule (∼100 to 220 kDa), which is bound to the ECM.
The large precursor molecule is cleaved in the ECM by proteases such as
plasmin to form the active TGF-β molecule (24 kDa) and a TGF-β-binding
protein. Local acidic environments, such as those induced by osteoclasts
during bone resorption, also activate TGF-β and allow it to be involved in
the bone remodelling process.
All isoforms of TGF-β bind to the same dimeric receptor complex that
consists of two receptor types. Binding of TGF-β ligands to the Type II TGFβ receptor (75–85 kDa) induces association of this molecule with the 50–
60 kDa Type I receptor, forming an activated heteromeric receptor complex
that consists of Type II and Type I polypeptides. The TGF-β receptor is a serine/threonine kinase. It catalyses the phosphorylation of serine and threonine residues in the receptor itself and in downstream cytoplasmic
messenger molecules.
The cytoplasmic mediators of the TGF-β family of receptors are called
Smads, messenger molecules present in organisms from worms and fruit
flies to mammals. There are nine vertebrate Smads resulting from three
types of Smad genes: receptor-activated, common and inhibitory.
Receptor-activated Smads are phosphorylated by the receptor kinase in
response to ligand binding. Smads 2 and 3 are the major receptor-activated
messengers for activin and TGF-β, respectively. Smads 1, 5 and 8 mediate
BMP receptor actions. After they are phosphorylated by the TGF-β receptor, the receptor-activated Smads associate with the common Smad
(Smad4) in the cytoplasm, forming a heterodimer that is translocated to the
nucleus where it alters the transcriptional activity of specific genes. The
third type of Smad, the inhibitory or anti-Smads, inhibits signalling by the
receptor-activated and common Smad complex. Smad6 and Smad7 are
inhibitory Smads for TGF-β, activin and BMP signalling. Unlike conventional transcription factors that bind directly to promoter sequences in
genes, the Smad molecules bind to other transcription factors that are
already bound to specific gene promoter sites. Interaction of Smads with
other transcription factors at the gene level provides a cooperative modulation of specific gene expression.
Most cells express TGF-β and have receptors for TGF-β, indicating the
importance of the autocrine function of TGF-β. In addition, TGF-β induces
the expression of its own gene, prolonging TGF-β secretion and enhancing
its autocrine effects. There are large quantities of TGF-β in platelets and bone
tissue where they regulate normal physiological processes. Unlike many
growth factors, low levels of TGF-β1 are always present in the circulation,
although the definitive site of production of this endocrine TGF-β1 is
unknown. Plasma levels of TGF-β1 are elevated in response to injury when
it is released from platelets involved in clot formation. TGF-β2 and TGF-β3
are not present in plasma and act at local sites within tissues as autocrine and
paracrine regulators of metabolism.
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TGF-β inhibits the growth of most epithelial, endothelial and
haematopoietic cell lineages. The inhibitory effects of TGF-β on cell growth
are antagonistic to those of typical mitogenic growth factors such as FGF and
PDGF. TGF-β has many effects on the ECM of bone, a function that is related
to tissue repair and remodelling. TGF-β increases the expression of ECM
proteins, such as fibronectin, collagen and decorin, while suppressing the
secretion of enzymes involved in matrix degradation, such as collagenase
and elastase.
In the skeletal system TGF-β isoforms act as growth and differentiation
factors (GDF) that regulate tissue differentiation and morphogenesis, bone
resorption and bone fracture repair. TGF-β is found in abundance in bone
and is produced and secreted by osteoblasts and chondrocytes, which also
respond to TGF-β in an autocrine manner. TGF-β and the Type I and Type II
TGF-β receptors are expressed during the early embryonic differentiation of
both endochondral and intramembranous bone. TGF-β is an important
molecular signal that induces the differentiation of mesenchymal cells into
chondrocytes and osteoblasts. Low levels of the three TGF-β isoforms are
expressed in mesenchymal cells, and expression of the isoforms by these
cells increases as mesenchymal cells form condensations in the areas of
future endochondral bone formation. The same increased expression of
TGF-β is seen in intramembranous bone mesenchymal cells that will differentiate into the osteoblasts.
In general, the physiological role of TGF-β in bone is to expand the progenitor cell population, increasing the pool of potential osteoblasts. TGF-β is
mitogenic for fibroblasts, undifferentiated mesenchymal cells, osteoblast
progenitors and osteoblasts. The mitogenic effects of TGF-β are similar to
those of PDGF, in that the predominant TGF-β effects are to increase the
number of bone precursor cells. Other growth factors and hormones regulate
differentiation of this expanded population of progenitor cells into mature
osteoblasts and osteocytes. Osteoblasts secrete TGF-β1 and TGF-β2 into the
bone matrix, where concentration of TGF-β1 is about eightfold higher than
TGF-β2. Transgenic mice that overexpress TGF-β2 in preosteoblasts have an
increased number of preosteoblasts, osteoblasts and osteocytes. They are
also characterized by increased bone turnover, both deposition and resorption. Although TGF-β can induce differentiation of bone marrow stromal
cells into osteoblasts, the inhibitory effects of TGF-β on differentiation predominate. The stimulatory effects of PTH and vitamin D3 on osteoblast differentiation are inhibited by TGF-β. This is reflected by the TGF-β inhibition
of the expression of osteoblast alkaline phosphatase and osteocalcin in
response to these hormones.
TGF-β is also produced by and has effects on the osteoclasts of the skeletal system, regulating the differentiation, activation and function of these
bone-resorbing cells. Although there are conflicting views about whether
TGF-β stimulates or inhibits osteoclast differentiation and bone resorption,
this is likely due to dose-dependent effects. Low doses of TGF-β (picogram
level), which are likely to be seen in physiological situations, stimulate osteoclast differentiation and bone resorption, while higher doses (nanogram
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range), which are not likely to be seen in the normal animal, inhibit osteoclast formation. During bone resorption, the acid and proteolytic enzymes
secreted by osteoclasts cleave and activate the large latent forms of TGF-β in
the ECM. Active TGF-β is then able to stimulate osteoclast differentiation by
at least two mechanisms that involve osteoblasts. Firstly, TGF-β stimulates
osteoblasts to secrete CSF-I. CSF-I is a growth factor that is believed to stimulate the proliferation and differentiation of osteoclast stem cells into osteoclasts. In addition, TGF-β stimulates osteoblasts to activate the
RANK/RANKL system (Chapter 4) that activates osteoclasts through their
cell-to-cell contacts with osteoblasts. This results in a stimulation of osteoclast differentiation and activation.
Bone morphogenetic proteins
The BMP are members of the TGF-β superfamily, discussed above.
Historically, the BMPs have been known by a variety of names, including
osteogenic protein (OP), cartilage-derived morphogenetic protein (CDMP)
and GDF. This array of terminology indicates the biological effects of BMP in
bone, cartilage and extraskeletal tissues. The current nomenclature of BMP
is perhaps misleading, as this family of peptides has many effects outside
the skeletal system. It is now known that BMPs induce a variety of cell functions, including stimulation of proliferation, differentiation, morphogenesis
and organogenesis of many systems. Their roles are not restricted to bone
development, and the BMPs might be more appropriately termed general
GDFs. The activity associated with BMPs in the skeletal system was first discovered in the 1960s by showing that extracts of demineralized bone could
induce bone formation de novo when implanted in ectopic sites, such as muscle, in rats. Ectopic treatment of non-skeletal tissues with BMP induces all of
the stages of embryonic bone formation, including the initial colonization
with undifferentiated mesenchymal cells that differentiate into chondrocytes,
followed by osteoblast and osteoclast differentiation and bone formation.
The BMPs were first cloned and characterized biochemically in the
1980s. The BMPs share several structural features with other TGF-β family
members. The BMPs are dimeric molecules, with two polypeptide chains
bound covalently with disulphide bonds. Like TGF-β, the BMPs are secreted
as large precursor proteins that must be proteolytically cleaved before they
become biologically active. The BMP family of peptide factors now encompasses at least 15 different members, a large proportion of the known TGF-β
superfamily.
The receptor system and cytoplasmic signalling pathways for the BMPs
are essentially identical to that of the TGF-βs, discussed above. The transmembrane BMP receptors are serine/threonine kinases that are activated
upon ligand binding, form dimers consisting of Type I and Type II receptors,
and undergo autophosphorylation. The Type I BMP receptor subunit catalyses the phosphorylation of the cytoplasmic signalling molecules, the Smads.
Smads 1, 5 and 8 are the receptor-activated molecules that form heteromers
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with the common Smad, Smad4, and mediate the signal transduction pathways of the BMPs. Smads 6 and 7 are anti-Smads that inhibit BMP, TGF-β
and activin signalling.
The BMPs are expressed early in embryonic development and can be
detected in day 6.5 mouse embryos; at a time that gastrulation (formation of
the three germ layers) is occurring. They are widely expressed and appear in
the extraembryonic membranes, such as the amnion, chorion and allantois,
of the developing fetus. They are also expressed in the cardiovascular system, specifically the myocardium, mesenchymal condensations of ribs, vertebrae and in the eye and tooth primordia. Later in development they appear
in chondrocytes of long bone and digits. The expression of the various isoforms of the BMPs is widespread and changes with developmental stage and
tissue type. Their expression is often localized in areas of epithelial–mesenchymal tissue interactions, leading to the idea that BMPs may be involved
in signalling between these embryonic tissues types.
In the adult, BMPs are believed to be involved in bone remodelling and
resorption. Bones are in a continuous state of turnover or remodelling in the
adult. This bone turnover involves osteoblasts and osteoclasts that respond
to systemic, local and mechanical signals. The BMPs are present in the bone
matrix and released at the sites of fracture. They are synthesized and
secreted by osteoblasts in vivo and in vitro. Upon release, the BMPs are
thought to induce mesenchymal stem cell commitment into the preosteoblast/osteoblast lineage. As mature osteoblasts secrete BMP, the initial
induction of increased osteoblast cell numbers by BMP acts as a positive
feedback pathway for BMP production, increasing BMP secretion from the
enhanced populations of osteoblasts. During bone fracture repair, BMP is
released at the site of fracture. All bone cells involved in fracture repair have
BMP receptors and respond to BMP. Specifically, BMP2 and BMP4 released
from the matrix or osteoblasts act on cells in the vicinity or recruits progenitor cells from the bone marrow to the fracture site where they differentiate
into osteoblasts that modulate new bone formation and fracture repair.
References and Further Reading
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Conover, C.A. (2000) Insulin-like growth factors and the skeleton. In: Canalis, E.,
(ed.) Skeletal Growth Factors. Lippincott,
Williams & Wilkins, Philadelphia,
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Derynck, R., Zhang, Y. and Feng, X.H.
(1998) Smads: transcriptional activators of TGF-beta responses. Cell 95,
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Gilboa, L., Nohe, A., Geissendörfer, T.,
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(2000) Bone morphogenetic protein
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cells: a new oligomerization mode for
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Molecular Biology of the Cell 11,
1023–1035.
Calcium Homeostasis and Regulation of Bone Growth
Givol, D. and Yayon, A. (1992) Complexity of
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Globus, R.K., Patterson-Buckendahl, P. and
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7
Hormones, Growth Factors
and Skeletal Muscle
Regulation of skeletal muscle growth and differentiation involves a plethora
of factors, including intrinsic genetic controls and extracellular signals, such
as morphogens, growth factors and hormones, that stimulate the developing
cells via receptors and nuclear transcription factors. The outline of myogenesis and the cells involved in myogenesis were described in Chapter 4. The
purpose of the current chapter is to examine the molecular mechanisms that
drive the processes of skeletal muscle growth, differentiation and function.
The size and numbers of myofibres are directly related to muscle mass
in the postnatal animal. During myogenesis, myoblasts proliferate, differentiate and fuse to form multinucleated myotubes and mature myofibres.
Cellular proliferation and differentiation during myogenesis are separate
and exclusionary events. After proliferation, myoblasts withdraw from the
cell cycle and fuse with one another to form myofibres that are no longer
capable of mitosis. In mammals, the numbers of myofibres are determined
during the latter part of gestation, during the last 10% to 20% of gestation in
cattle and pigs. The size of the myofibres is determined by a number of factors, including genetics, nutrition, exercise and the endocrine environment.
Experimental Systems to Study Myogenesis
The molecular signals that induce the myogenic pathway during embryogenesis have been discovered only within the last few years, owing to the
development of extremely sensitive molecular biological tools. Much of the
information on embryonic development and its regulation at the molecular
level has been produced using experimental models such as the round
worm, Caenorhabditis elegans, and the fruit fly, Drosophila melanogaster. The
use of these small, relatively simple invertebrates with short lifespans (2 to
3 weeks) and well-characterized genetics and developmental processes have
led to fundamental discoveries of the molecular mechanisms involved in
development of organ systems that hold true for almost all animals.
Findings from these animals have been corroborated in vertebrate species
such as the chicken and mouse embryo. These animals are used because of
their abundance, low cost and short embryonic and adult lifespan. Many of
the proteins that act as intercellular signals and intracellular transcription
146
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Hormones, Growth Factors and Skeletal Muscle
147
factors are essentially identical in species that range from round worms to
humans. This conservation of the structure and function of gene products
from widely disparate organisms points to the essential nature of these biochemical signals and their central role in developmental processes that
determine the form and function of the mature organism.
As we have seen, the formation of skeletal muscle in birds and mammals
begins near the embryonic neural tube with the formation of pluripotent
somites. These paired structures, aligned along the presumptive backbone of
the embryo (embryonic neural tube), give rise to the dermis of the skin, the
axial skeleton (ribs, vertebrae) and most of the animal’s skeletal muscle,
including the body, back and limb musculature. Formation of skeletal muscle in the limbs involves the delamination of muscle progenitor cells from
the somite followed by their migration to the limb bud. In the limb bud, the
progenitor cells undergo proliferation to increase precursor cell numbers
and then fuse to form the mature multinucleated muscle fibres. Each of these
events is strictly controlled by intrinsic genetic mechanisms and external
signals.
Myogenic cell systems
In addition to studies using intact embryos to examine early muscle development, many of the effects of growth factors and hormones on skeletal
muscle cell growth and differentiation are studied using isolated cells
in vitro. Use of these cells eliminates the effects of multiple complex interactions between hormones, growth factors and nutrients that occur in the
intact animal. Cells are grown in a buffered culture medium containing glucose, vitamins and minerals in an appropriate osmotic balance. Temperature,
pH, oxygen and carbon dioxide are also maintained at constant levels. This
allows one to study the effect of external factors in a controlled environment.
Culture medium is often supplemented with serum, which is added to maintain cell survival and induce mitosis. Unfortunately, serum contains variable
and unknown quantities of hormones, growth factors, vitamins and minerals. These unknowns contribute to the variation of data produced from this
system. On the other hand, cells can be grown in a serum-free, defined culture medium that provides known amounts of nutrients and growth factors.
Various combinations of albumin (as a protein and osmotic source), insulin,
the iron-binding protein transferrin, glucocorticoids and thyroid hormones
may be added in order to maintain normal cell metabolism and substitute
for the lack of serum in defined media.
The use of cells maintained in an artificial environment in vitro to study
physiological processes suffers from some drawbacks. Most obviously, cells
grown in a single layer on a flat Petri dish lack the normal architecture seen
in the animal. The three-dimensional relationship of the cells in the original
tissue and the interaction of myogenic cells with non-muscle cells such as
adipocytes, fibroblasts and vasculature are absent. The intimate cellular connection with ECM, which we now know is an important source of local
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growth factors and regulatory molecules, is lost in cell cultures. Contact with
nerve cells, which play an important role in muscle growth, development
and function, is also missing. Although cell cultures are a valuable tool to
study growth factor effects on myogenesis, results must be interpreted with
these limitations in mind.
Several types of muscle cell cultures are used to study myogenesis
in vitro. Primary cultures of myogenic cells, derived from embryonic, fetal
and postnatal muscle from a variety of species, have been used to study
myogenesis. These cultures have a limited lifespan and, in general, cell isolates are prepared fresh for each experiment. While this may introduce individual animal variation into the experiment, many scientists believe that the
cells used in primary cell cultures are more similar to those of the animal
from which they are isolated. Myogenic cell lines are generally ‘immortalized’ by transformation to provide cells that grow continuously. Many cell
lines are derived from cancerous cells that continuously replicate. Thus, cell
lines often represent aberrant, continuously proliferating cell behaviour,
respond differently to hormones and growth factors than non-transformed
cells and express a different set of genes than non-transformed cells.
Nevertheless, several myogenic cell lines have been used to study muscle
development and these have provided insight into the regulatory mechanisms involved in myogenesis. These include the L6 myoblast cell line,
derived from fetal rat thigh muscle, as well as clones from L6 cells, L6A1 and
L6E9. The C2C12 clone of the C2 satellite cell line is derived from the adult
mouse, as are the cell lines Sol8, from the soleus muscle, and the BC3H1 cell
line.
Primary cell cultures and cell lines are used to study the processes of
proliferation, differentiation and fusion of myogenic cells. Cell proliferation
can be studied by the direct enumeration of cell numbers using a microscope
or electronic counting, or by quantifying the incorporation of radioactive
thymidine (either 14C or 3H) into DNA. Differentiation of myoblasts into
myotubes (multinucleated myofibre precursors) can be measured microscopically, counting the number of multinucleated cells formed in response
to a stimulus, or molecular markers of the differentiated state can be examined. The activity of the muscle-specific creatine kinase enzyme or the
expression of the myosin mRNA or protein are often used as markers for
muscle differentiation. New molecular biology methods are used to quantitate the changes in gene expression during differentiation of MRFs such as
MyoD or myogenin.
Muscle Regulatory Factors (MRFs)
MRFs are transcription factors that induce commitment to the myogenic lineage and induce myofibre differentiation. Each MRF can induce the complete myogenic programme in non-skeletal muscle cell types, such as
fibroblasts in vitro, resulting in the formation of skeletal muscle. The MRFs
belong to the superfamily of over 400 bHLH transcription factors. In verte-
Hormones, Growth Factors and Skeletal Muscle
149
brates, the MRF subfamily consists of four members: Myf5 (myogenic factor
5), MyoD (myogenic determination factor D), MRF4 and myogenin. When
these transcription factors are activated, they form homodimers with themselves and heterodimers with other bHLH transcription factors called E proteins. After dimerization, the MRFs bind to nucleotide sequences in
the promotor region of affected genes called Eboxes, with the sequence
5′-GAGCTG-3′. Binding of MyoD and Myf5 to E boxes induces local changes
in the chromatin (chromatin remodelling) in the regulatory region of genes.
This results in the activation of specific gene targets by MyoD and Myf5.
The MRFs are expressed sequentially during embryonic development.
They appear first in the somites and later in myoblasts of the limb bud. The
formation of somites during embryonic development occurs in an anterior to
posterior fashion. Similarly, the development of the embryonic anterior
(forelimb) limb buds occurs before the hind limbs. Accompanying these
developmental events, MRF expression is first seen in the anterior somites
and as development progresses, expression of MRFs is seen in the posterior
somites. Likewise, the anterior limb buds express the MRFs before the posterior limb buds. The Myf5 gene is the first MRF to be expressed (Table 7.1).
Myf5 occurs in the epaxial dermomyotome, where it is found first, at day 8
in the mouse embryo (21-day gestation). It is expressed throughout the
entire myotome as development proceeds and is downregulated by day 10.5.
Myogenic cells in the mouse limb bud express Myf5 between embryonic
days 10 and 12.
Expression of MyoD begins at day 9.75 in the hypaxial portion of the
somite and is seen throughout the somite by day 11 of development. Somitic
expression of MyoD is maintained throughout the fetal life. Like Myf5,
MyoD is expressed between days 10 and 12 in the limb bud. Myogenin
expression in the somites begins at day 8.5 and continues throughout fetal
Table 7.1. MRF gene expression in mouse embryos.
MRF gene
Embryonic source
Myf5
Somite
Limb bud
Myogenin
Somite
Limb bud
MRF4
Somite
Myofibre
MyoD
Somite
Limb bud
Embryonic day (21 days
gestation)
8–10.5
10–12
8.5–21
10.5–21
9–11.5
16–21
9.75–21
10–12
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development. In the limb buds, myogenin expression begins at day 10.5.
Myogenin gene expression ceases in the postnatal animal.
The gene expression of MRF4 occurs in a biphasic manner during two
embryonic periods. It is first expressed from day 9 to day 11.5 in the somites, is
downregulated, and then is re-expressed in differentiated muscle fibres beginning at day 16 and for the remainder of development. Expression of MRF4 persists in the postnatal animal, where it is the most abundant of the MRFs.
Effects of the MRFs on muscle development
The functions of the MRFs have been determined using knockout mice in
which a specific MRF gene (or genes) is disrupted. The expression of MyoD
and Myf5 provides an excellent example of biological redundancy during
skeletal muscle differentiation. When the MyoD gene is deleted from
embryos, the resulting animals are viable, able to reproduce and have no
obvious skeletal muscle defects. Expression of myogenin and MRF4 is unaffected. Closer examination of the development of these MyoD-deficient mice
showed that the migration of hypaxial myoblasts, but not epaxial myoblasts,
was delayed by about 3.5 days. In addition, the expression of Myf5 mRNA
is increased in the postnatal mouse in the absence of MyoD. These observations suggest that in the absence of MyoD, Myf5 expression fills a compensatory role that allows normal muscle development to proceed even in the
absence of MyoD. On the other hand, when the Myf5 gene is deleted, the
embryos are not viable, the distal portion of the ribs does not form and
somite formation is delayed for 2 days. These events occur despite the observation that both MyoD are myogenin are expressed normally. No obvious
skeletal muscle defects are observed in neonatal mice after the deletion of
Myf5 gene and the expression of other MRFs is not altered. In contrast to the
MyoD-deficient animals, in which hypaxial myoblast migration is delayed,
the formation of epaxial musculature is delayed in Myf5 knockout mice.
When both the Myf5 and MyoD genes are deleted, myoblasts and skeletal
muscle are not formed. The neonatal mice are immobile and die immediately
after birth.
Mice lacking the myogenin gene die soon after parturition. The animals
have major skeletal muscle deficiencies throughout the body, reduced myofibre density and muscle mass, as well as spinal and rib deformities. The muscles primarily affected by the absence of myogenin are those of the hypaxial
musculature, in which muscle fibres are scarce or absent. The numbers of
myoblasts in myogenin knockout mice are equivalent to those in normal
mice. Studies in vivo and in vitro demonstrate that myogenin expression is
upregulated during myoblast differentiation and fusion. Together, these
observations suggest that the primary effects of myogenin occur after
myoblast formation and that myogenin is required for the terminal differentiation of myoblasts into myotubes and myofibres.
The effects of deletion of the MRF4 gene are complicated by its proximity to the Myf5 gene. These genes are contiguous in mice and deletion of the
Hormones, Growth Factors and Skeletal Muscle
151
MRF4 gene also affects the regulation of the Myf5 gene. This is because regulatory elements that affect the regulation of the Myf5 gene are scattered
throughout the introns in the MRF4 and Myf5 gene complex. MRF4 knockouts die in the perinatal period with deficient epaxial myogenesis, intercostal muscles and rib anomalies. A defect in myotome formation is also seen
between days 9 and 11, when MRF4 is first expressed during normal development. Expression of the other three MRFs is also reduced at day 10, but
myogenesis is apparently normal by day 14. Postnatal expression of myogenin is increased in the MRF4 knockout mice, suggesting that myogenin
may, in part, compensate for the absence of MRF4.
These elegant experiments demonstrate the importance of the MRFs in
skeletal muscle development. MyoD and Myf5 play essential roles in the
recruitment, migration and differentiation of the myogenic lineage. The lack
of overt skeletal muscle defects in single gene knockouts indicates that the
effects of MyoD and Myf5 are redundant and in the absence of one of these
MRFs, the other one can compensate for its loss. In the absence of both factors, no myoblasts or skeletal muscle is formed. For these reasons, MyoD
and Myf5 are considered to be the primary myogenic regulatory factors that
are required for the determination of the myogenic lineage. Myogenin and
MRF4 are secondary factors in the regulation of myogenesis, as their effects
occur later in development. Myogenin acts in myoblasts to induce terminal
differentiation while MRF4 affects mature myofibres in the embryo and
postnatal animal. In addition, observations from the MyoD and Myf5 knockout mice suggest that MyoD and myogenin regulate epaxial muscle development and the combination of Myf5 and MRF4 regulates hypaxial muscle
formation.
Although the primary actions of the MRFs are during the embryonic
development of muscle, they may also have effects in the adult animal. In
postnatal muscle, MRF4 is the most abundant of the MRFs, suggesting a possible role of MRF4 in the maintenance of the differentiated state of skeletal
muscle. MyoD and myogenin expression in mature skeletal muscle occurs
only at low levels. In adult rats, MyoD expression is restricted to fast glycolytic
fibres while myogenin is found only in slow oxidative fibres. This suggests
that MyoD and myogenin may have fibre-specific effects in adult animals.
Regulation of MRF expression
The expression of MRFs is regulated by several factors (Fig. 7.1). The earliest
processes in the formation of specialized myogenic precursor cells in the
somites and their subsequent migration are accompanied by the increased
expression of a number of genes. The expression of MRFs in the somites is
influenced by paracrine signals from adjacent tissues. For example, the
embryonic notochord and the ventral neural tube secrete SHH, while morphogens from the wingless genes (wnts 1, 3 and 4) are secreted from the dorsal neural tube and ectoderm of the embryo. Along with SHH, wnt initiates
dermomyotome formation and myogenic cell recruitment, by inducing
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Fig. 7.1. Regulation of embryonic myogenesis by MRFs and morphogens.
MyoD and Myf5 and myogenin expression in the differentiating muscle
cells. SHH induces the expression of Myf5 and MyoD in the dermomyotome
and rapid differentiation and hypertrophy of skeletal muscle.
In the somites, the development of a muscle progenitor pool in the lateral dermomyotome is accompanied by the increased expression of the transcription factor Pax3 (Paired Box 3). Pax3 is first expressed at day 8.5 in the
dermomyotome. The expression of Pax3 continues in the myoblasts migrating from the somite to the limb bud. Embryonic mice in which the Pax3 gene
is deleted die before day 15 of development. In these animals, myogenic cells
do not migrate from the somites to areas of muscle formation and there is an
absence of muscles in the limbs, back and diaphragm. The expression of
MyoD is under the dual control of Pax3 and Myf5. Although MyoD expression is not affected in the absence of Pax3, ablation of both Pax3 and Myf5
genes abolishes MyoD expression and skeletal muscle does not form.
Another transcription factor, Lbx1 (Ladybird homeobox 1), is involved in
the delamination of lateral somite cells and is required for migration of
somitic cells into the limb bud. Lbx1 is first expressed at day 8 in the dermomyotome and Lbx1 is found in migrating cells at day 10. Ablation of the
Lbx1 gene leads to an absence or extreme reduction of limb muscles, as cells
from the somite fail to migrate. In addition, HGF, and its tyrosine kinase
receptor called c-Met, are involved in the migration of cells from the lateral
somites to the limb buds. HGF is discussed in detail below.
Upon arrival in the limb bud, the myoblasts rapidly proliferate under
the influence of FGF-2, -4 and -8, which are secreted by the ectoderm overlying the limb bud. Proliferation of the myoblasts increases cell numbers to
provide additional sources of myoblasts while differentiation is delayed. The
mesenchyme in the posterior margin of the limb bud, called the zone of
polarizing activity, secretes the SHH, which is responsible for the anterior–posterior positioning of the limb and induction of myogenic factors that
stimulate differentiation. Both FGF and SHH are required to coordinate limb
formation and in the absence of either factor limb development ceases.
Hormones, Growth Factors and Skeletal Muscle
153
Growth Factors Affecting Muscle Growth
IGFs
The IGFs play a central role in the development, differentiation and maintenance of skeletal muscle (Fig. 7.2). The IGFs are produced by the liver as well
as by cells of the skeletal muscle system, including myoblasts, satellite cells,
myofibres and fibroblasts. Thus, these ubiquitous growth factors may act via
endocrine, paracrine and autocrine pathways to affect skeletal muscle
growth and metabolism. Current experimental data suggest that the effects
of IGFs on muscle growth are primarily local autocrine and paracrine effects.
With the exception of the C2C12 satellite cell line, there are few or no muscle GH receptors and, when studied in vitro, GH effects on muscle growth
and differentiation cannot be demonstrated. This suggests that GH has no
direct anabolic effects on myoblasts, satellite cells or myotubes and the
observed effects of GH in the intact animal are probably due to locally produced IGFs. In skeletal muscle, the IGFs increase glucose and amino acid
uptake, decrease proteolysis and increase protein synthesis. In addition, they
have effects on muscle precursor cells, increasing myoblast and satellite cell
proliferation and differentiation. The IGFs are the only extracellular growth
factors which are capable of inducing the terminal differentiation of
myoblasts and satellite cells. The direct effects of IGFs on mature myofibres
result in an induction of muscle hypertrophy of glycolytic, Type IIb fibres.
Most primary cell cultures and myogenic cell lines secrete and respond
to IGFs, suggesting an autocrine mechanism of action. In addition, embryonic and adult muscle cells express IGFs during development. In porcine
embryos, the expression of muscle IGF-I and IGF-II genes increases during
the latter phase of primary muscle fibre formation, from days 44 to 59 of gestation. During the time of secondary myofibre formation (day 75 and on) in
pigs, expression of IGF-I is increased further while IGF-II expression steadily
declines. These observations suggest that the IGFs play important regulatory
roles in all phases of myogenesis in vivo and in vitro.
Administration of IGFs to experimental animals induces an increase in
muscle mass while the removal of IGF reduces muscle mass. Knockout mice
in which the IGF-I gene is deleted results in a phenotype similar to myogenin knockout mice, with a generalized failure of skeletal muscle development. Similarly, Type I IGF receptor knockout mice are about half the size of
wild-type mice and suffer from generalized muscle hypoplasia. Deletion of
the Type II IGF receptor results in neonatal mice that are actually about 30%
heavier than their normal littermates. These mice have elevated levels of
IGF-II and die in the postnatal period. This suggests that the role of the Type
II receptor is not to mediate the effects of IGF-II on muscle growth. Instead,
this receptor is believed to act as a negative regulator of circulating IGF-II
levels, internalizing IGF-II into cells and degrading it. This protects the animal from excessively high levels of IGF-II.
Studies on myogenic cells in vitro show that the IGFs induce both proliferation and differentiation of myoblasts and satellite cells. Both IGF-I and
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IGF-II are capable of myogenic regulation, but larger doses of IGF-II are
required to induce the same effects as those seen with IGF-I. This supports
the belief that the effects of IGF-II on muscle cells (as well as other cells) are
mediated by the Type I IGF receptor or, possibly, the insulin receptor. As IGFII has a lower affinity for these receptors than their primary ligands, higher
concentrations of IGF-II are needed to induce muscle functions.
Nevertheless, IGF-II may be more important than IGF-I for differentiation of
skeletal muscle. IGF-II levels increase 20–30-fold during differentiation of
myoblasts into myotubes, suggesting that autocrine effects of IGF-II are
involved in skeletal muscle differentiation.
The effects of IGFs on myogenesis depend upon the stage of myoblast
development. At early times of development, the IGFs stimulate proliferation. Sustained stimulation of cells by the IGFs leads to cellular differentiation, characterized by activation of myogenin, Myf5 and MyoD expression.
In addition, the effects of IGFs on muscle cells are concentration-dependent.
Low concentrations of IGF-I or IGF-II stimulate dose-dependent differentiation, while higher concentrations inhibit differentiation of myoblasts.
These effects, seen in the artificial environment of cell culture in vitro, likely
mimic physiological processes in the animal. Thus, a muscle cell is likely to
be exposed to IGFs only during certain developmental stages that are regulated by specific, temporal IGF gene expression. Likewise, very high, localized concentrations of IGFs may be expected to be present in the ECM.
Exposure of cells to these elevated levels as paracrine or autocrine signals
is likely to induce different responses than seen with lower concentrations
of IGFs.
Two different Type I IGF receptor-activated intracellular pathways
mediate the effects of IGFs in myogenic cells. The effects of IGF-I on cell proliferation is believed to be mediated by the MAP kinase pathway while the
PI-3 kinase pathway mediates the effects of IGF-I on muscle differentiation.
Likewise, the IGFs have differential effects on the expression of the MRF
genes during cell proliferation and differentiation. When cell proliferation is
induced by the IGFs, MRF expression is inhibited and when myogenic differentiation is induced by the IGFs, MRF4 and myogenin gene expression are
upregulated. When the myogenin gene mRNA is neutralized with antisense
oligonucleotides, IGF-I treatment fails to induce differentiation. This indicates that myogenin expression is an obligatory event in the IGF-I induction
of differentiation. When the Type I IGF receptor is deleted, myogenin and
MyoD expression are delayed in vivo and in vitro, suggesting that activation
of the IGF receptor is at least partially responsible for the induction of these
genes. Thus, the IGFs play a central role in the induction of proliferation and
differentiation of myogenic precursor cells, acting to induce or suppress
MRF expression. These effects of IGFs result in an increase in myoblast and
myofibre numbers.
In addition to the effects of IGFs on myogenic cell proliferation and differentiation, the IGFs play crucial roles in the induction and maintenance of
mature skeletal muscle growth. Muscle hypertrophy is stimulated in transgenic mice in which the IGF-I gene is specifically expressed in skeletal muscle.
Hormones, Growth Factors and Skeletal Muscle
155
Transfection of myotubes with the IGF-I gene in vitro also induces cellular
hypertrophy. In the intact animal, adult skeletal muscle growth is due to the
addition of satellite cell nuclei to the amitotic myofibres. Several studies in
multiple laboratories over the past 20 years have shown that the IGFs
induce satellite cell proliferation, differentiation and fusion to form
myotubes.
Effects of the IGFs on hypertrophy are accompanied by alterations in
protein and carbohydrate metabolism. The IGFs stimulate amino acid
uptake and protein synthesis while inhibiting protein degradation in a variety of myogenic cells in vitro, including myoblasts, satellite cells and
myotubes. Treatment of mice with IGF-I also stimulates protein synthesis
and inhibits protein degradation in skeletal muscle. The metabolic energy
that is used for muscle hypertrophy, protein synthesis and contraction is
derived from the oxidation of glucose. One of the early bioassays for IGFs
involved the IGF-stimulated oxidation of glucose by isolated adipose tissue.
In muscle cells, IGFs also stimulate glucose uptake and oxidation.
Myostatin
Myostatin, also known as GDF-8, is a secreted growth factor that is a member of the TGF-β superfamily. Myostatin is a 15 kDa peptide with autocrine
and paracrine actions in skeletal muscle. The myostatin molecule is highly
conserved between a wide range of species, and is identical in mice, rats,
humans, pigs and birds. This molecular conservation suggests that myostatin is an evolutionarily conserved essential factor that plays a central role
in physiological processes that are common to a wide spectrum of animals.
Myostatin is expressed in cells very early in embryonic development and is
present in chick blastoderm, the avian equivalent of the mammalian blastocyst. It is also expressed later in embryogenesis, when it appears in the dermomyotome of the somites. In addition, myostatin is expressed in the adult,
and is found in satellite cells and mature myofibres. Myostatin is not
restricted to skeletal muscle, and is also present in adipose tissue and the
mammary gland.
In contrast to many of the growth and transcription factors that regulate
myogenesis, myostatin is a negative regulator of muscle growth. Although
many growth factors delay differentiation by increasing proliferation, the
TGF-β superfamily of peptides, including BMP, TGF-β and myostatin,
reduce cell proliferation rates and, thus, delay differentiation. Myostatin
inhibits myogenic differentiation by inhibiting the expression of the myogenic transcription factors MyoD and myogenin. When myoblasts
are treated with myostatin, cell proliferation, DNA and protein synthesis are
reduced. In contrast, when muscle atrophy is induced by denervation, the
synthesis of myostatin protein and mRNA is upregulated in the muscle
fibres. This indicates that myogenin plays a significant role in the activation
and recruitment of satellite cells that are responsible for the regeneration of
the atrophied muscle. The myostatin gene is expressed in proliferating satellite
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cells in vitro, and its expression increases prior to myotube formation. Thus,
myostatin may be a major factor that activates satellite cells during muscle
hypertrophy and regeneration. This is further supported by studies in
knockout mice, in which deletion of the myostatin gene stimulates satellite
cell division, skeletal muscle hypertrophy and a two- to threefold increase in
muscle mass.
A naturally occurring mutation of the bovine myostatin gene has been
identified that results in an inactive myostatin molecule. This has been shown
to be the cause of the muscle hypertrophy that results in the so-called doublemuscled phenotype in cattle breeds. Although these animals do not have
twice the number of muscles as other breeds, they are characterized by excessive musculature due to hypertrophy. Cattle breeds such as the Belgian Blue,
Piedmontese and Asturiana de la Valle result from mutational inactivation of
the myostatin molecule. Interestingly, a human infant has recently been identified with a myostatin mutation. This child displays the excessive musculature and muscular hypertrophy characteristic of myostatin deficiency.
FGF
FGF is a potent inhibitor of muscle differentiation but plays a key role in early
myoblast development. FGF has both autocrine and paracrine effects on
developing muscle cells. The FGF gene is expressed in the early embryonic
somite and later in development in the overlying ectoderm of the limb bud.
After synthesis and secretion, FGF is stored in the ECM, where it is bound to
heparin sulphate and Syndecan3. FGF mRNA and protein are found in proliferating satellite cells, myoblasts and macrophages. FGF receptors are present
in myoblasts and satellite cells, but are absent in myotubes. FGF inhibits differentiation by stimulating mitosis of myoblasts and satellite cells, thereby
delaying differentiation. In proliferating myogenic cells, FGF suppresses the
expression of the MRFs, MyoD and myogenin. This inhibits MRF induction of
the specific myogenic genes needed for muscle differentiation. FGF increases
chemotaxis of stem cells and stimulates angiogenesis, the formation of new
blood vessels for tissue growth. FGF inhibits the expression of IGF-II and may
block some of the effects of IGF-II on myogenic differentiation. If the effects of
FGF are blocked in myoblasts by receptor mutations, myoblasts differentiate
prematurely into myofibres. As a result, there is a 30% reduction in limb muscle weight and a 50% reduction in muscle fibre numbers in these animals. This
demonstrates the importance of FGF in myogenesis, a factor that has an
inhibitory effect on muscle differentiation, but one that is essential to provide
adequate numbers of myoblasts to form normal musculature.
TGF-β
Like FGF, TGF-β is a potent inhibitor of muscle differentiation. Many cells produce TGF-β, including myoblasts and regenerating muscle tissue. After secre-
Hormones, Growth Factors and Skeletal Muscle
157
tion, TGF-β is stored in the ECM bound to the protein decorin. The main isoform of TGF-β found in skeletal muscle is TGF-β3. It has been shown that TGFβ inhibits the differentiation of myogenic cells. It does this by slowing the
proliferation of myoblasts and satellite cells. As was the case with FGF, TGF-β
treatment inhibits the expression of MyoD and myogenin in myogenic cells. In
addition TGF-β3 has distinct effects on the ECM, inducing the synthesis of collagen and fibronectin. Overall, TGF-β is an important inhibitor of the terminal
differentiation of skeletal muscle, while ensuring that there is an adequate precursor pool of undifferentiated, quiescent myogenic cells. Coincidentally,
TGF-β induces angiogenesis of developing tissues, providing the vascular
supply needed for muscle growth and development.
BMPs
The BMPs, members of the TGF-β family, have distinct effects during skeletal muscle differentiation. In the embryo, cells of the ectoderm, mesoderm
and the neural tube produce the BMPs that act on adjacent myogenic cells in
the somites and the limb bud. BMP4 functions in mesodermal cell determination into the myogenic line and formation of myoblasts. Like other members of the TGF-β family, BMP4 slows myoblast proliferation and prevents
premature myoblast differentiation in the somites.
Hepatocyte growth factor
HGF was initially characterized as a potent mitogen for hepatocytes. It is
identical to ‘scatter factor’, named because of its stimulatory effects on
epithelial cell motility. It has since been shown to be mitogenic for many
endothelial and epithelial cells. HGF is an autocrine and paracrine mediator
of morphogenesis in the myogenic cell lineage and is also important in the
development of the liver and placenta. The HGF molecule consists of a disulphide-linked heterodimer with a molecular weight of 70 to 80 kDa. HGF is
secreted as a large monomer that is activated by proteolytic removal of a portion of the precursor molecule. Secreted HGF is stored in the ECM of skeletal muscle. Heparin sulphate of the ECM is required for the HGF activation
of the HGF receptor. The effects of HGF are mediated by the cell surface
tyrosine kinase receptor, c-Met.
HGF plays a central role in the initial events that trigger the myogenic
cascade. HGF induces the initial conversion of epithelial cells in the somite
into mesenchymal cells, establishing the somitic pool of myogenic precursor
cells. This epithiomesenchymal conversion is accompanied by the assumption of mesenchymal cell motility and migration of myoblasts from the
somite to areas of hypaxial muscle formation. Along with the transcription
factors Lbx1 and Pax3, HGF mediates myoblast delamination from the dermomyotome and myoblast migration to sites of muscle formation. The
delamination and migration of myoblasts from the somite to the developing
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limb bud is stimulated by paracrine HGF, which is secreted by mesenchymal
cells along the routes of myoblast migration and by limb bud mesenchyme.
When HGF or the HGF receptor (c-Met) gene is inactivated, hypaxial
myoblast migration fails, and skeletal muscles in the limb and diaphragm do
not form, although epaxial skeletal muscle is not affected. As this migratory
failure occurs despite continued Lbx1 expression, HGF and its receptor are
believed to be the primary inducers of myoblast delamination, migration
and colonization of sites of muscle formation.
HGF has also been shown to be involved in adult muscle hypertrophy
and regeneration. Muscle regeneration in response to physical trauma or
exercise essentially recapitulates the embryonic myogenic pathway.
Quiescent satellite cells are activated, proliferate, differentiate and fuse into
contractile myotubes in vitro. In animals, satellite cells fuse with myofibres
and provide new nuclei at local sites for muscle repair and hypertrophy.
HGF is the first growth factor to activate satellite cell proliferation, ending
the dormant state and inducing satellite cell entry into the cell cycle and
mitosis. HGF is synthesized and secreted by primary cultures of chicken and
turkey satellite cells and the C2 satellite cell line, and HGF is restricted to
satellite cells and the surrounding ECM. HGF is not detected in musclederived fibroblasts or myofibres. As satellite cells express the c-Met HGF
receptor, HGF is believed to act via an autocrine pathway to activate quiescent satellite cells. Expression of the c-Met receptor in satellite cells declines
during differentiation. HGF effects on satellite cells are mediated by an inhibition of expression of the bHLH transcription factors, MyoD and myogenin.
The regulation of myogenesis by growth factors is shown in Fig. 7.2.
Hormones regulating muscle growth
The role of hormones that directly regulate the growth and development of
skeletal muscle is relatively limited. As discussed in detail in the current and
preceding chapters, GH plays an important, albeit indirect, role in the development of skeletal muscle, with its actions mediated by the IGFs. As discussed in previous chapters, the steroids and the catecholamine-derived
β-agonists provide important tools in the regulation of farm animal muscle
growth. Other hormones, such as insulin, thyroid hormones and glucocorticoids, play a primarily permissive role in the growth, differentiation and
maintenance of muscle.
Insulin is required to maintain adequate cellular energy supply, by
maintaining a constant concentration of serum glucose and by regulating
cellular glucose uptake, an essential component of normal cell growth and
metabolism. In the animal, insulin stimulates the deposition of glycogen,
protein and triglycerol, acting in concert with other anabolic and catabolic
hormones. Insulin has no effects on brain glucose uptake and the gut and
skin are largely insensitive to insulin. Insulin also stimulates amino acid
transport, protein synthesis and inhibits protein degradation during tissue
growth. Although insulin is essential for protein accretion, insulin treatment
Hormones, Growth Factors and Skeletal Muscle
159
Stem cells – pluripotent mesoderm
↓
Determination: HGF, IGF, SHH
Myoblasts and satellite cells (SCs activated by HGF/c-Met)
↓
Determination: HGF
↓
Proliferation: FGF, PDGF, TGF-β (−), IGF
↓
Differentiation: IGFs, myogenin/MRF4
↓
Fusion: IGF, HGF
Myotubes
↓
Fusion, hypertrophy : IGF, FGF, HGF
Myofibres
Fig. 7.2. The role of growth factors in the regulation of myogenesis.
of animals does not produce positive results, due to insulin induction of
hypoglycaemia. In postnatal skeletal muscle, insulin has anabolic effects.
Individuals with diabetes mellitus suffer from muscle wasting, a condition
that is reversed by insulin treatment. The effects of insulin on growth and
differentiation of myogenic cells requires very high, pharmacological doses
of insulin. For example, insulin induces growth and differentiation of satellite cells, myoblasts and fibroblasts when 1 µM insulin is present. This is an
extremely high dose of insulin, equivalent to ∼5 mg/ml, a concentration
never achieved in the animal. The effects of insulin on myogenic growth and
differentiation are probably due to cross-reaction with the Type I IGF receptor at high concentrations of insulin.
Adrenal glucocorticoids are generally considered to inhibit growth.
Glucocorticoids are considered as glucose-sparing hormones that reduce
glucose use by tissues, increase gluconeogenesis and induce hyperglycaemia. These effects are in opposition to those seen with insulin, and glucocorticoids provide a balance to the effects of insulin on glucose
metabolism. Physiological doses of glucocorticoids inhibit cell replication
and protein synthesis in a variety of cell types in vitro. In vivo, glucocorticoid
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Chapter 7
treatment of animals slows growth and inhibits the secretion of GH and the
IGFs. Protein catabolism by muscle is increased by glucocorticoids, in vivo
and in vitro. This results in a reduced incorporation of amino acids into muscle protein and an increased release of amino acids by muscle. Long-term or
high-dose treatment of animals induces muscle atrophy. Nevertheless, glucocorticoids, in the form of synthetic dexamethasone, are often added to
myogenic cell culture medium. In the artificial environment in which cells are
grown in vitro, the presence of glucocorticoids provides a counter-regulatory
effect for the anabolic actions of other factors (e.g. insulin) to ensure adequate
balance between intracellular and extracellular glucose homeostasis.
It has long been known that a deficiency of thyroid hormones, either
from a dysfunctional thyroid or an absence of dietary iodine, leads to growth
retardation. Thyroid hormones play important roles in the development of
the CNS and neonatal and pre-pubertal deficiencies in thyroid hormones
result in reduced CNS myelination, mental retardation and the syndrome
known as cretinism in humans. Thyroid hormones are largely permissive
agents for muscle growth and differentiation. Along with GH and growth
factors, thyroid hormones are required for normal muscle development.
Thyroid hormones stimulate the synthesis of protein and RNA in skeletal
muscle and are important in maturation of skeletal muscle. As neonatal rat
muscle matures, myosin shifts from a slow, embryonic isoform to a
fast twitch, adult isoform. This transition is accelerated by thyroid hormones
and is retarded in hypothyroid animals. The primary metabolic effect of thyroid hormones is to stimulate energy use, oxidative metabolism and metabolic rate. Exposure of animals to high doses of thyroid hormones induces
protein degradation and muscle atrophy. The effects of thyroid hormones on
energy metabolism and muscle atrophy, which predominate in long-term or
high-dose treatments, preclude the use of thyroid hormones as growth
promotants.
Further Reading
Allen, R.E. and Boxhorn, L.K. (1989) Regulation
of skeletal muscle satellite cell proliferation
and differentiation by transforming
growth factor-beta, insulin-like growth
factor I, and fibroblast growth factor.
Journal of Cellular Physiology 138, 311–315.
Allen, R.E., Sheehan, S.M., Taylor, R.G.,
Kendall, T.L. and Rice, G.M. (1995)
Hepatocyte growth factor activates quiescent skeletal muscle satellite cells in vitro.
Journal of Cellular Physiology 165, 307–312.
Atchley, W.R. and Fitch, W.M. (1997) A natural
classification
of
the
basic
helix–loop–helix class of transcription
factors. Proceedings of the National
Academy of the Sciences USA 94,
5172–5176.
Borycki, A.G. and Emerson, C.P. Jr. (2000)
Multiple tissue interactions and
signal transduction pathways control
somite myogenesis. Current Topics in
Developmental Biology 48, 165–224.
Hormones, Growth Factors and Skeletal Muscle
Borycki, A.G., Brunk, B., Tajbakhsh, S.,
Buckingham, M., Chiang, C. and
Emerson, C.P. Jr. (1999) Sonic hedgehog
controls epaxial muscle determination
through Myf5 activation. Development
126, 4053–4063.
Brand-Saberi, B. (ed.) (2002) Vertebrate myogenesis. In: Results and Problems in Cell
Differentiation, Vol. 38. Springer-Verlag,
New York, 242 pp.
Brohmann, H., Jagla, K. and Birchmeier, C.
(2000) The role of Lbx1 in migration of
muscle precursor cells. Development 127,
437–445.
Florini, J.R. and Ewton, D.Z. (1988) Actions of
transforming growth factor-beta on muscle cells. Journal of Cellular Physiology 135,
301–308.
Florini, J.R., Ewton, D.Z., Falen, S.L. and
Van Wyk, J.J. (1986) Biphasic concentration
dependence of stimulation of myoblast
differentiation
by
somatomedins.
American Journal of Physiology 250,
C771–C778.
Florini, J.R., Ewton, D.Z., Magri, K.A. and
Mangiacapra, F.J. (1994) IGFs and
muscle differentiation. In: LeRoith, D.
and Raizada, M.K. (eds) Current
Directions in Insulin-like Growth Factor
Research. Plenum Press, New York,
pp. 319–326.
Hasty, P., Bradley, A., Morris, J.H.,
Edmondson, D.G., Venuti, J.M., Olson,
E.N. and Klein, W.H. (1993) Muscle deficiency and neonatal death in mice with a
targeted mutation in the myogenin gene.
Nature 364, 501–506.
Kablar, B., Krastel, K., Ying, C., Tapscott, S.J.,
Goldhamer, D.J. and Rudnicki, M.A.
(1999) Myogenic determination occurs
independently in somites and limb buds.
Developmental Biology 206, 219–231.
Kaul, A., Koster, M., Neuhaus, H. and Braun,
T. (2000) Myf-5 revisited: loss of early
myotome formation does not lead to a
rib phenotype in homozygous Myf-5
mutant mice. Cell 102, 17–19.
Kocamis, H. and Killefer, J. (2002) Myostatin
expression and possible functions in animal muscle growth. Domestic Animal
Endocrinology 23, 447–454.
161
Milasincic, D.J., Calera, M.R., Farmer, S.R.
and Pilch, P.F. (1996) Stimulation of
C2C12 myoblast growth by basic fibroblast growth factor and insulin-like
growth factor 1 can occur via mitogenactivated protein kinase-dependent and
-independent pathways. Molecular and
Cellular Biology 16, 5964–5973.
Molkentin, J.D. and Olson, E.N. (1996)
Defining the regulatory networks for
muscle development. Current Opinion in
Genetics and Development 6, 445–453.
Oksbjerg, N., Gondret, F. and Vestergaard, M.
(2004) Basic principles of muscle development and growth in meat-producing
mammals as affected by the insulin-like
growth factor (IGF) system. Domestic
Animal Endocrinology 27, 219–240.
Olson, E.N., Arnold, H.H., Rigby, P.W. and
Wold, B.J. (1996) Know your neighbors:
three phenotypes in null mutants of the
myogenin bHLH gene MRF4. Cell 85, 1–4.
Olwin, B.B., Bren-Mattison, Y., Cornelison,
D.D.W., Federov, Y.V., Flanagan-Steet, H.
and Jones, N.C. (2002) Role of cytokines in
skeletal muscle growth and differentiation.
In: Sassoon, D.A. (ed.) Advances in
Developmental Biology and Biochemistry.
Vol. 11. Stem Cells and Cell Signalling in
Skeletal Myogenesis. Elsevier, Amsterdam,
pp. 97–126.
Palmer, C.M. and Rudnicki, M.A. (2002) The
myogenic regulatory factors. In: Sassoon,
D.A. (ed.) Advances in Developmental
Biology and Biochemistry, Vol. 11. Stem Cells
and Cell Signalling in Skeletal Myogenesis.
Elsevier, Amsterdam, pp. 1–32.
Rawls, A. and Olson, E.N. (1997) MyoD
meets its maker. Cell 89, 5–8.
Rios, R., Carneiro, I., Arce, V.M. and Devesa,
J. (2002) Myostatin is an inhibitor of
myogenic differentiation. American
Journal of Physiology. Cell Physiology 282,
C993–C999.
Rudnicki, M.A., Schnegelsberg, P.N., Stead,
R.H., Braun, T., Arnold, H.H. and
Jaenisch, R. (1993) MyoD or Myf-5 is
required for the formation of skeletal
muscle. Cell 75, 1351–1359.
Tajbakhsh, T. and Buckingham, M. (2000) The
birth of muscle progenitor cells in the
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mouse: spatiotemporal considerations.
Current Topics in Developmental Biology
48, 225–268.
Valdez, M.R., Richardson, J.A., Klein, W.H.
and Olson, E.N. (2000) Failure of Myf5 to
support myogenic differentiation without myogenin, MyoD and MRF4.
Developmental Biology 219, 287–298.
Webb, S.E. and Lee, K.K. (1997) Effect of
platelet-derived growth factor iso-
forms on the migration of mouse
embryo limb myogenic cells. The
International Journal of Developmental
Biology 41, 597–605.
Zhu, X., Hadhazy, M., Wehling, M.,
Tidball, J.G. and McNally, E.M. (2000)
Dominant negative myostatin produces hypertrophy without hyperplasia in muscle. FEBS Letters 474,
71–75.
8
Hormones, Growth Factors
and Adipose Tissue
This chapter is devoted to the control of growth and differentiation of adipose tissue by hormones and growth factors. Like bone, adipose tissue is
classified histologically as a connective tissue. In contrast with most organs,
adipose tissue is present not in a single location, but is spread throughout the
body in many sites. White adipose tissue is an energy-dense tissue that
serves as a nutrient depot for the body, a tissue that stores energy in the form
of triacylglycerols in times of excess caloric intake. In times of nutrient deficiency lipid reserves stored in white adipose tissue are mobilized to provide
metabolic energy to the animal. In contrast, brown adipose tissue is a specialized type of fat involved with cold adaptation and the generation of heat
to maintain body temperature. This chapter deals primarily with white adipose tissue. Recently, adipose tissue has been shown to be a dynamic tissue
that secretes a variety of factors that are actively involved in the regulation
of such diverse processes as appetite, metabolism, the immune response,
reproduction and haematopoiesis. These newly discovered functions
expand the role of adipose tissue to that of an endocrine organ that secretes
a variety of hormones and growth factors. The fundamental differentiation
and development of adipose have been examined in Chapter 4. The primary
goal of this chapter is to examine the hormones, growth factors and transcription factors that control adipose tissue development and maintain adipose tissue function. In addition, the role of adipose tissue as an endocrine
organ will be discussed.
Cell Systems Used to Study Adipogenesis
Because of the diffuse nature of adipose tissue depots and the difficulties
with examining a small population of undifferentiated preadipocytes in the
whole animal, most studies examining the differentiation of adipocytes are
performed using enriched preadipocyte cell preparations in vitro.
Preadipocytes are present in the stromovascular cell fraction of adipose tissue and primary cell cultures derived from the stromovascular cell fraction
can be used to study differentiation into adipocytes. The stromovascular
©K.L. Hossner 2005. Hormonal Regulation of Farm Animal Growth (K.L. Hossner)
163
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fraction of adipose tissue contains fibroblasts, endothelial cells and stromal
cells. These cells cannot be distinguished morphologically or biochemically
from the presumptive preadipocytes in the stromovascular fraction, but may
provide paracrine agents and a cellular environment more akin to the
situation in vivo. The presence of preadipocytes in primary cultures can be
distinguished from other cell types only by post hoc analysis. That is, only
after differentiation and the expression of differentiation-specific molecular
markers or formation of the lipid-containing adipocyte phenotype can the
presence of the precursor preadipocyte cell be deduced.
The use of primary cell cultures of adipocytes, like those of any tissue,
has the advantage that the cells are isolated from ‘normal’ animals using the
species of choice. This is very important when studies about adipocyte
metabolism from farm animals are undertaken, as these cells are available
only from isolated primary cell cultures. Primary tissue culture cells are not
transformed into immortalized cells capable of continuous replication
in vitro and they are thus believed to be more similar to the cells seen in the
intact animal than those of cell lines. A disadvantage of using primary cells
is that the cells do not survive over a long period of time in vitro, and must
be isolated anew for each experiment. This involves a great deal of labour
and expense and introduces individual animal variation into the analysis.
An alternative to using primary cell cultures is the use of established
preadipocyte cell lines. Pluripotent mouse fibroblast cell lines, such as the
C3H10T1/2 and the NIH 3T3 cell lines, are capable of differentiation into
preadipocytes and adipocytes. Clones of the mouse embryonic NIH 3T3
fibroblasts, denoted 3T3-L1 and 3T3-F442A, are preadipocyte cell lines that
are committed to the adipogenic lineage and undergo differentiation into
adipocytes under appropriate hormonal treatment. These cell lines have provided invaluable tools to study adipose cell differentiation and provided
much of our current knowledge about the cellular and molecular regulation
of adipogenesis. The advantages of using cell lines are that they can be cultured easily and grown continuously in vitro. On the other hand, as these
cells are usually derived from embryonic or tumourigenic sources, they may
not display the physiological characteristics or responses to stimuli that
affect primary cell cultures isolated from normal animals. The 3T3 cell lines
also suffer from aneuploidy, a state in which chromosome numbers are not
an exact multiple of the original chromosome number of the animal from
which they are derived. This may alter their physiological characteristics
and responses to external stimuli. Cells grown in monolayers in vitro also
suffer from a lack of three-dimensional organization seen in the animal and
lack their normal extracellular support and interactions with surrounding
lipogenic and non-lipogenic cells. The available cell lines differentiate into
white adipose tissue. Cell lines for the study of brown adipose tissue are not
available. In addition, available cell lines are usually derived from human or
rodent sources and may have no relevance to domestic animal physiology.
Preadipocyte cell lines for specific species, such as ruminants, do not exist.
The process of 3T3-L1 preadipocyte differentiation follows an ordered,
repeatable sequence of events that can be reliably studied in vitro (Fig. 8.1).
Hormones, Growth Factors and Adipose Tissue
165
Fig. 8.1. The differentiation of 3T3-L1 cells.
Most of our knowledge of the processes involved with adipocyte differentiation, gene expression and hormonal regulation of these processes has been
gained from this system. Similar processes occur when preadipocytes isolated from stromovascular cells are studied in vitro. After cells are put into
culture, they are maintained in a growth medium supplemented with mammalian serum. This provides the cells with nutrients and growth factors
needed to induce a mitotic state and cells undergo proliferation. After the
cells have replicated to the point of confluence, that is, when they cover the
entire culture plate, they exit the cell cycle and become quiescent. They are
then transferred to a culture medium supplemented with a hormone cocktail that induces differentiation into adipocytes during a 4-day period.
Preadipocyte differentiation is stimulated by a medium containing a hormonal mixture of insulin or IGF-I, a glucocorticoid such as dexamethasone
and isobutylmethylxanthine (IBMX). IBMX is a chemical that inhibits phosphodiesterase, the enzyme responsible for cAMP breakdown. Thus, the
addition of IBMX increases cellular cAMP concentrations, and in the presence of dexamethasone and insulin, induces differentiation into lipid-filled
adipocytes. The addition of this hormone cocktail induces the cells to reenter the cell cycle and undergo one or two rounds of mitosis. This renewed
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mitotic behaviour is called clonal expansion. After exposure to this hormonal cocktail for 2 days, it is replaced by a medium containing only insulin.
This induces the cells to withdraw from the cell cycle and begin the final
process of terminal differentiation into adipocytes. After the 4-day hormonal
induction period, more than 90% of the cells have assumed the adipocyte
phenotype.
As with any cell studied in vitro, preadipocyte cell replication can be
studied by measuring the incorporation of radioactive thymidine into
nuclear DNA or by counting the number of cells that result from an experimental treatment. More sophisticated analyses monitor the expression of cell
cycle-specific transcription factors to reflect the induction of mitosis.
Differentiation of preadipocytes into mature adipocytes involves changes in
the expression of over 2000 genes. The expression of some of these genes,
either as protein or mRNA, can be quantitated and used as markers for
assumption of the differentiated state. A few prominent molecular markers
that are used to assess the differentiated state in adipocytes include genes
expressed early in differentiation such as LPL, α-collagen VI and fatty acid
transporter. Later markers of adipocyte differentiation include leptin, glycerol-3-phosphate dehydrogenase (GPDH), FAS, lipid-binding protein (ap2)
and adipsin.
Transcription Factors and Adipogenesis
Cell differentiation is regulated by differential gene transcription. The differentiation of cells involves the shift of the gene expression pattern of cells
from those genes associated with the pluripotent state of the primitive progenitor cells to an expression pattern seen in the differentiated cells. This is
accompanied by morphological changes in the cell that arise as a consequence of altered gene expression. For example, during the differentiation of
adipocytes, cell morphology changes from an irregular fibroblast-like shape
to a cell that is rounded and begins to accumulate lipid. Changes in the hormonal and growth factor environment induce the expression of transcription
factor genes that induce changes in adipocyte-specific gene expression and,
subsequently, morphological and functional transformations.
Several transcription factors modulate adipose cell growth and differentiation. They are expressed sequentially in a time-specific, but overlapping
manner by differentiating preadipocytes and terminally differentiated
adipocytes. Many of them act in synergy with each other, enhancing one
another’s effects and inducing each other’s expression. The cell system used
most frequently to study these changes is the 3T3-L1 preadipocyte cell line,
described earlier. With few exceptions, this model system is the basis for the
following discussion of adipogenesis, as studied in vitro. The transcription
factors that drive adipocyte differentiation are not expressed during the initial cell proliferation and confluence induced by serum. The transcription
factors appear only after the addition of the hormone cocktail that reactivates the confluent cell cultures and initiates the differentiation programme
Hormones, Growth Factors and Adipose Tissue
167
of these cells. The transcription factors act at the level of the gene to induce
or repress the expression of specific genes that regulate adipocyte differentiation and maintain the differentiated state. There are three major families of
transcription factors that regulate adipocyte differentiation. These are peroxisome proliferator-activated receptor (PPAR), the C/EBP and the adipocyte
determination and differentiation factor 1/sterol regulatory element-binding protein 1 (ADD1/SREBP1).
PPARs
As their name implies, the PPARs were first discovered as gene products that
were induced by agents that stimulate peroxisome proliferation.
Peroxisomes are intracellular, membrane-bound organelles, similar to lysosomes, that are present in all plant and animal cells. They are abundant in
liver cells, where they occupy about 1% of the cell volume. Enzymes within
the peroxisomes use oxygen as an oxidizing source and produce hydrogen
peroxide (H2O2) as a by-product. In the liver, they play important roles in
amino acid, uric acid and ethanol oxidation. Peroxisomes are also very
important in the degradation of long-chain fatty acids via the β-oxidation
pathway. This results in the formation of acetyl-CoA and H2O2.
The PPAR family of transcription factors plays a central role in the
induction of the adipocyte differentiation programme. The potency of the
PPARs in induction of adipogenesis is illustrated by the observation that
PPARs can stimulate the differentiation of myoblasts into adipocytes and can
induce the commitment of pluripotent fibroblasts, such as the C3H10T1/2
cell line, into the adipogenic cell lineage. The PPARs belong to the nuclear
hormone receptor family. These receptors are activated by ligand binding to
form heterodimers with other nuclear hormone receptors, the retinol X
receptors (RXR). Formation of PPAR/RXR dimers induces binding of the
complex to specific DNA sequences in the PPAR response elements. The specific sequences are called the DR-1 site and these are present in the promoter
region of specific genes. Binding of the PPARs to the DR-1 sequences activates the transcription of genes that induce adipose differentiation and lipid
synthesis.
The PPARs exist as three isoforms, PPARα, PPARδ (also called PPARβ)
and PPARγ that share a high degree of amino acid homology in the two
molecular domains that bind DNA and ligands. Despite these close homologies, the PPARs display distinct ligand specificity and DNA-binding sites.
Ligands for the PPARs include long-chain fatty acids, fatty acid metabolites
and drugs like the thiazolidinediones (TZDs) that reduce circulating lipid
concentrations. The TZDs are some of the most important and widely studied of the PPARγ ligands. They are synthetic drugs that were developed for
treatment of Type II (insulin-resistant) diabetes, as they reduce hyperlipidaemia and increase insulin sensitivity. It was only later that these drugs
were found to be high affinity ligands for PPARγ and potent inducers of
adipocyte differentiation. Most naturally occurring ligands for PPARγ are
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Chapter 8
arachidonic acid derivatives such as prostaglandin D2 and the prostaglandin
J2 metabolite, 15-deoxy-∆12,14-prostaglandin J2.
The members of the PPAR family have different adipogenic activity,
based on their potencies to induce 3T3-L1 cell differentiation into
adipocytes. PPARα has only low adipogenic activity, PPARδ has no adipogenic activity while PPARγ is the most potent member of this family in the
induction of the adipogenic lineage. PPARα occurs only at low levels in
white adipose tissue but is present in high concentrations in brown adipose
tissue. The adipogenic role of PPARα in white adipose tissue is believed to
be minor and PPARα may be more important in the regulation of lipid
metabolism. Mice in which the PPARα gene is disrupted have normal adipose tissue development, but display reduced β-oxidation of lipids in peroxisomes. The second member of the PPAR family, PPARδ, is found in many
tissues in addition to adipocytes and its role, if any, in adipose cell differentiation is controversial and unclear.
PPARγ is the most potent of the PPAR family in the induction of
adipocyte differentiation. It is the master regulator of adipocyte differentiation and adipocyte metabolism. It is the most abundant of the PPARs, occurring in adipose at levels that are 30-fold higher than in other tissues. PPARγ is
the most fat-specific of the PPAR family. It is found in adipose tissue and
adipocyte cell lines, but absent or present only in low levels in other tissues.
Two forms of PPARγ, PPARγ1 and PPARγ2, result from alternate splicing of
mRNA and alternate promoter sites of the PPARγ gene. PPARγ1 expression
is widespread and is found in many different tissues, while PPARγ2 is found
mainly in adipocytes. This suggests that PPARγ2 may have a more important
role in adipocytes than PPARγ1, but this suggestion has not been adequately
explored.
Expression of the PPARγ gene occurs early in adipogenesis, beginning
about 1.5 days after hormone treatment of 3T3-L1 cells and persisting
throughout terminal differentiation (Fig. 8.2). Overexpression of the PPARγ
gene in preadipocytes induces the entire differentiated adipocyte phenotype, including the typical rounded adipocyte cell shape, lipid accumulation
and the development of insulin sensitivity. PPARγ-induced differentiation of
these cells is accompanied by the expression of adipocyte-specific genes such
as fatty acid-binding protein (ap2), LPL, acetyl-coenzyme A carboxylase, leptin, FAS and SCD-1. In addition to its role in adipogenesis, PPARγ is also
thought to be responsible for maintenance of the differentiated state of
adipocytes. PPARγ is expressed in mature adipocytes and the PPAR response
element is present in the genes for several lipogenic enzymes, including LPL,
PEPCK and SCD.
Ligands which bind to and activate PPARγ include arachidonic acid
metabolites, some polyunsaturated fatty acids (linoleic acid), the TZD drugs
and non-steroidal anti-inflammatory drugs (NSAIDs). The binding affinity
of the naturally occurring ligands for PPARγ is quite low, in the micromolar
range. This contrasts with the high affinity (nanomolar) of the TZDs for
PPARγ. The low affinity of currently identified PPARγ ligands casts doubt on
their role in the physiological activation of PPARγ and the identity of
Hormones, Growth Factors and Adipose Tissue
169
Fig. 8.2. Expression of transcription factors and adipocyte genes during differentiation of
3T3-L1 cells.
endogenous ligands for PPARγ is still uncertain. PPARγ is inactivated by
phosphorylation of specific amino acid residues in the PPARγ molecule.
Activation of the intracellular MAP kinase pathway inhibits adipogenesis.
This has been shown to be due to the phosphorylation of PPARγ and RXR,
which inhibits their activity.
C/EBPs
The C/EBP transcription factors are members of the basic leucine zipper
family of transcription factors. The C/EBPs form homo- or heterodimer
complexes with other molecules and bind to the CCAAT enhancer elements
of gene regulatory sequences. The C/EBP are not limited to fat cells. They
also play a role in the differentiation of hepatocytes and granulocyte cells of
blood. There are six isoforms of the C/EBP molecules, which are formed by
the use of differential transcription start sites on the C/EBP genes.
The primary C/EBP isoforms are C/EBPα, C/EBPβ and C/EBPδ.
C/EBPβ and C/EBPδ mRNA and protein appear in 3T3-L1 cells about 12 h
after hormone treatment and the expression of these genes subsides after
2 days (Fig. 8.2). The expression of C/EBPα begins after about 1.5 days of hormone treatment, just before terminal differentiation and the initiation of
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adipocyte-specific gene expression. C/EBPα expression persists in the differentiated adipocytes throughout the culture period.
The functional roles of the C/EBPs have been studied using transgenic
mice and cell lines and in mice in which the C/EBP gene has been disrupted.
Ectopic expression of the gene for C/EBPα or C/EBPβ in 3T3-L1
preadipocytes induces differentiation into adipocytes, while expression of
the C/EBPδ gene accelerates preadipocyte differentiation. If both genes for
C/EBPβ or C/EBPδ are disrupted in 3T3-L1 cells, there is a severe reduction
in adipose tissue differentiation. In mice in which the C/EBPβ or C/EBPδ
gene is disrupted, white adipose tissue is present at normal levels, but lipid
accumulation and UCP-1 in brown adipose tissue is reduced. When both
C/EBPβ and C/EBPδ genes are disrupted in mice, most of the animals die of
unknown causes in the perinatal period. Of the 15% that survive, there is an
extreme reduction in brown adipose tissue and a lesser reduction in white
adipose tissue. These observations suggest that C/EBPβ and C/EBPδ play
relatively modest roles in white adipocyte differentiation but are important
in the development of brown adipose tissue and lipid accumulation.
C/EBPα, along with PPARγ, plays a major role in the induction of
preadipocyte differentiation. C/EBPα is expressed at the same time as
PPARγ, initiated near the end of the mitotic phase of the 3T3-L1 differentiation programme, just before the expression of adipose-specific genes.
Transfection of preadipocytes with the C/EBPα gene blocks mitosis, and the
inhibition of the actions of C/EBPα with antisense RNA inhibits differentiation of preadipocytes. These observations suggest that the expression of
C/EBPα terminates the clonal expansion of preadipocytes and induces differentiation. When C/EPBα is expressed at the end of the mitotic period of
clonal expansion, adipocyte-specific gene expression is initiated, even in the
absence of hormonal inducers. Co-expression of C/EBPα and PPARγ in
fibroblasts enhances adipocyte formation while co-expression in myoblasts
provides a signal strong enough to transform the myoblasts into adipocytes.
C/EBPα expression is induced by PPARγ, while C/EBPα reciprocally
induces PPARγ expression. C/EBPα and PPARγ act synergistically to induce
adipocyte differentiation. In addition, C/EBPα gene expression is autoregulated by the C/EBPα protein. This occurs via interaction with C/EBP
response elements in the promoter region of the C/EBPα gene. The primary
function of C/EBPα is believed to be maintenance of the differentiated state
of adipocytes.
ADD1/SREBP1
The ADD1/SREBP1 transcription factor plays a dual role in adipocyte
metabolism. It stimulates adipocyte differentiation and is a key factor in the
cholesterol-regulated activation of the transcription of adipocyte-specific
lipogenic genes. The cumbersome dual nomenclature for this molecule is
due to the independent characterization of ADD1 in rodents and SREBP1 in
humans, but its use conveys the dual function of ADD1/SREBP1 in
Hormones, Growth Factors and Adipose Tissue
171
adipocyte differentiation and lipid metabolism. ADD1/SREBP1 belongs to
the bHLH family of transcription factors that includes the myogenic regulatory factors that mediate muscle development. ADD1/SREBP1 is stored
within the cell as an inactive molecule bound to the membranes of the endoplasmic reticulum. Upon activation, ADD1/SREBP1 is proteolytically
cleaved and released from the endoplasmic reticulum. The activated molecule binds to two different sites in the promoter regions of target genes:
sterol response elements (SRE) and the typical bHLH sites, the E-box motifs.
This dual specificity accounts for the effects of ADD1/SREBP1 on lipid
metabolism and adipocyte differentiation.
The presence of ADD1/SREBP1 is required for adipocyte differentiation.
However, in contrast to PPARγ, ADD1/SREBP1 alone cannot induce the
entire adipogenic programme. When the gene for SREBP1 is disrupted in
mice, most of the animals die before parturition. Surviving animals have
normal fat levels. Overexpression of ADD1/SREBP1 in pluripotent fibroblasts induces some fibroblasts to differentiate into adipocytes, but only
under conditions that are strongly permissive for differentiation, such as the
presence of the hormone cocktail used to stimulate adipocyte formation. In
the presence of hormonal inducers of differentiation, overexpression of the
ADD1/SREBP1 gene in 3T3-L1 preadipocytes increases adipocyte differentiation markers and lipid accumulation compared to hormone treatment
alone.
The expression of ADD1/SREBP1 is induced in 3T3-L1 preadipocytes
about 12 h after addition of the hormone cocktail that stimulates the differentiation programme. Its expression continues throughout clonal expansion,
growth arrest and terminal differentiation of the adipocytes. The timing of
ADD1/SREBP expression precedes and overlaps that of PPARγ expression.
Thus, ADD1/SREBP1 may induce PPARγ expression and, as ADD1/SREBP1
and PPARγ genes are co-expressed, their actions appear to be synergistic.
This synergism is seen in 3T3-L1 preadipocytes transfected with the
ADD1/SREBP1 and PPARγ genes. When both genes are present, there is an
increase in the adipocyte-specific transcriptional activity over that seen in
cells singly transfected with only the ADD1/SREBP1 or PPARγ gene. This
synergistic effect is due to ADD1/SREBP1 effects on PPARγ activity.
ADD1/SREBP1 is believed to enhance the activity of PPARγ by two separate
mechanisms. First, the lipogenic effects of ADD1/SREBP1 may stimulate the
synthesis of natural ligands that activate PPARγ. Secondly, ADD1/SREBP1
may directly activate transcription of the PPARγ gene.
In addition to its role in adipogenesis, ADD1/SREBP1 regulates genes
for fatty acid, triglycerol and cholesterol metabolism. Enhanced expression of
ADD1/SREBP1 is associated with induction of genes associated with
increased lipid and cholesterol metabolism, such as glycerol phosphate acyltransferase, LPL and FAS genes. The expression of this transcription factor is
regulated by nutrients in mature animals. Under conditions in which lipogenesis is stimulated, as in refeeding after a fasting period, ADD1/SREBP1
expression is increased. Insulin is a potent regulator of ADD1/SREBP1 in adipose tissue and the liver. The elevated levels of insulin that follow nutrient
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ingestion mediate the effects of nutrient induction of ADD1/SREBP1 expression. Conversely, ADD1/SREBP1 expression is reduced during fasting. A
summary of the relationships between the transcription factors that regulate
adipogenesis is presented in Fig. 8.3.
Nuclear co-activators and co-repressors: fine-tuning the adipogenic response
The effects of transcription factors on gene transcription are regulated by
other nuclear factors called co-activators and co-repressors of transcription.
Co-activators of PPARγ and the C/EBPs bind to these transcription factors
and enable the maximum activation of gene transcription. The interaction of
multiple co-activators with the primary transcription factors at different
times adds an additional level and a ‘fine-tuning’ of the regulation of
adipocyte gene transcription. Co-activators of gene expression enhance gene
transcription by altering chromosomal chromatin proteins, exposing gene
transcription sites on DNA that are usually covered by tightly bound histones and other proteins. Nuclear co-activators also recruit components of
the transcriptional apparatus to the gene transcription site.
Fig. 8.3. Interrelationships and regulation of adipocyte transcription factors.
Hormones, Growth Factors and Adipose Tissue
173
Co-activators of PPARγ bind to the carboxyl terminus of these nuclear
hormone receptors in the presence of ligand. A complex of co-activators,
p160/CBP/p300, bind to PPARγ and attract other activating molecules. The
p160/CBP/p300 complex has histone acetyltransferase (HAT) activity. The
HAT activity enzymatically acetylates histone protein in chromatin, inducing conformational changes and opening specific gene sites for efficient
transcription. The second class of ligand-dependent co-activators is the
DRIP/TRAP/ARC complex. Important isoforms of this complex, the proteins DRIP205 and TRAP220, are vital regulatory molecules that share amino
acid homologies with a PPARγ-binding protein (PBP).
PPARγ co-activator-1 (PGC-1) is a ligand-independent co-activator of
PPARγ that has no HAT activity. Instead, PGC-1 interacts with the
p160/CBP/p300 complex that provides HAT activity. PGC-1 is restricted to
brown adipose tissue, where its expression is induced by cold stress and
alterations in nutrient intake. In brown adipose tissue, PGC-1 stimulates
mitochondrial biogenesis and the expression of UCP-1 and components
of the cytochrome chain. PGC-2 is also a ligand-independent co-activator
of PPARγ. Like PGC-1, PGC-2 has no HAT activity, but may recruit other
co-activators with HAT activity to the PPARγ complex.
C/EBPβ interacts with two types of nuclear co-activators. The
SWI/SNF complex induces chromatin remodelling in certain cells. As we
have seen, the CBP/p300 complex is a strong activator of PPARγ. This
complex also activates C/EBPβ. This cross-reactivity of CBP/p300 with
PPARγ and C/EBPβ may, in part, explain the synergistic relationship
between these two transcription factors in the induction of adipocyte
differentiation.
Co-repressors of transcription factors usually bind to the unliganded
nuclear receptor and are released upon ligand binding to the receptor. A
co-repressor of PPARγ, called Sirt 1 (sirtuin 1), binds to and represses genes
activated by PPARγ. Sirt 1 is the mammalian homologue of a molecule that
has deacetylase activity. Deacetylase activity may reverse histone acetylation
catalysed by co-activators, returning histones to their original binding sites
on specific genes and inhibiting gene transcription. Overexpression of the
Sirt 1 gene is 3T3-L1 cells interferes with adipogenesis and antisense RNA
inhibition of Sirt 1 enhances adipogenesis. In differentiated adipocytes,
upregulation of Sirt 1 stimulates lipolysis and reduces fat content.
Hormones and Growth Factors Affecting Preadipocytes
and Adipocytes
Insulin
Insulin plays an essential role in stimulating both proliferation and differentiation of preadipocytes. In addition, insulin is a major endocrine regulator
of lipogenesis and the maintenance of the differentiated state of mature
adipocytes. The main effects of insulin on preadipocytes are to stimulate
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differentiation of these cells into adipocytes. Inclusion of IGF-I or pharmacological levels of insulin in the hormone cocktail used to stimulate differentiation of 3T3-L1 cells is required for preadipocyte differentiation. In primary
cell cultures of preadipocytes derived from the stromovascular fraction of
adipose tissue, the inclusion of insulin at physiological levels enhances morphological differentiation and the expression of lipogenic enzymes and their
mRNAs. The presence of glucocorticoids, usually in the form of dexamethasone, and GH enhance insulin action on adipocyte differentiation. On the
other hand, pharmacological levels of insulin (10 µg/ml) induce mitosis in
preadipocytes. This effect is likely mediated by cross-reaction of insulin with
the IGF-I receptor or induction of IGF-I synthesis and secretion by
adipocytes.
In the mature adipocyte and adipose tissue, insulin acts as a lipogenic
hormone, increasing energy storage in the form of triglycerol. Insulin
induces the expression of several lipogenic enzymes, including LPL, FAS
and acetyl-coenzyme A carboxylase. The receptor-mediated effects of
insulin on lipogenic enzymes are mediated by increased expression of the
transcription factors, ADD1/SREBP1, which, in turn, induces PPARγ
expression.
GH and the IGFs
The effects of GH on preadipocytes have been studied extensively. While the
GH receptor is present in preadipocytes and adipocytes, the effects of GH on
adipocyte metabolism, when studied in vitro, are variable and hard to reproduce. Some of the conflicting results are due to the differences in metabolism
between cell lines and primary cultures of preadipocytes. When primary cultures of preadipocytes are studied, they are isolated from adipose tissue
using proteolytic enzymes. Exposure of the cells to these enzymes may alter
surface-bound GH receptor concentration or function. This may explain the
differential effects of GH seen in freshly isolated vs longer-term incubations.
GH effects also depend upon the species or cell lines used. This is especially
important when one wishes to study farm animal adipocyte growth and differentiation, as primary cell cultures must be used. There are few or no cell
lines available for domestic animal studies. In addition, GH effects can vary
according to the depot source of fat studied. Different depot sites of adipose
tissue display different metabolism and responses to external factors. All GH
effects require serum at low levels in vitro, suggesting that other serumborne factors provide a synergistic factor to allow the GH to have an effect
on these cells.
Many studies with GH use preadipocyte cell lines and the results in
these cells often do not coincide with studies in primary cultures of cells
derived from the stromovascular fraction. For example, treatment of
preadipocytes in primary culture with GH does not induce differentiation.
In preadipocyte cell lines, such as the 3T3-L1 and 3T3-F442A cells, however,
physiological levels of GH promote differentiation into adipocytes. GH stimu-
Hormones, Growth Factors and Adipose Tissue
175
lates proliferation of primary cultures of stromovascular cells, but inhibits
proliferation of 3T3-F442A cells. These stimulatory effects of GH on proliferation and differentiation on cells in primary culture are probably mediated
by IGF-I. When primary preadipocytes are treated with GH, IGF-I synthesis
and secretion are induced. If the effects of IGF-I are inhibited by neutralization with antibodies to IGF-I, proliferation and differentiation are blocked.
This provides strong evidence that the effects of GH on preadipocyte proliferation and differentiation are mediated by IGF-I in an autocrine or
paracrine manner.
In mature adipocytes treated with GH in vitro, GH has differential acute
and chronic effects on lipid metabolism. Acute effects are seen in adipose tissue that has been deprived of GH for several hours. These effects are seen in
hypophysectomized animals and in cell cultures. Acute exposure of cells to
GH induces transient insulin-like, anabolic effects on lipid metabolism,
including increased glucose transport and oxidation, increased lipogenesis
and reduced lipolysis. After prolonged (1–2 h) exposure of GH in vitro, typical GH effects are seen and lipogenesis is reduced. Often, GH induces lipolysis in vitro. As discussed in Chapter 5, GH treatment of animals in vivo
induces lipolysis only under conditions of energy restriction. The reduction
in fat mass induced by GH treatment is due to an inhibition of lipogenesis,
not the stimulation of lipolysis, when an adequate diet is available. The frequent observation that GH is lipolytic in vitro suggests that the cells are in an
inadequate nutritional state that allows GH to induce lipolysis. The inhibition of lipogenesis by GH is reflected by the effect of GH on lipogenic
enzymes. GH treatment reduces the activity of both FAS and LPL and antagonizes the insulin induction of FAS expression.
Adipocytes and preadipocytes synthesize and secrete IGFs and their
binding proteins. In rats, the synthesis of IGFBP-2 and -3 is restricted to
preadipocytes, while IGFBP-5 is secreted by both preadipocytes and mature
adipocytes. GH regulates the synthesis of IGF-I in adipose tissue. Adipose
IGF-I mRNA is stimulated by GH treatment in vivo and by GH treatment of
preadipocytes in vitro. Hypophysectomy of rodents suppresses adipose tissue IGF-I mRNA. The genes for the IGFs are expressed at low levels in
preadipocytes in vitro and their expression increases during differentiation
into mature adipocytes. In stromovascular cell preparations, IGF-I enhances
both proliferation and differentiation of preadipocytes. This is reflected by
increased expression of enzyme markers of adipocyte differentiation such as
α-GPDH and LPL. Low levels of insulin and glucocorticoids act synergistically to enhance the effects of IGF-I on differentiation. IGF-I in serum-free
media induces differentiation in 3T3-L1 and primary cultured preadipocytes
of most species studied.
In mature adipocytes studied in vitro, IGF-I has insulin-like effects. IGFI treatment increases lipogenesis, glucose uptake and oxidation, and the synthesis and secretion of LPL. As high doses of IGF-I are required for these
responses, these effects of IGF-I are thought to be mediated by IGF-I interactions with the insulin receptor. In vivo treatment of animals with IGF-I
results in a slight or no increase in body fat.
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Glucocorticoids
Glucocorticoids are major permissive hormones that are required for the
optimal physiological processes of most cells and tissues. The most revealing effects of glucocorticoids on adipose tissue are seen in the clinical syndrome known as Cushing’s disease. In individuals with this syndrome,
excessive ACTH secretion stimulates excess adrenal glucocorticoid release
and elevates circulating glucocorticoid concentrations. A typical phenotype
associated with Cushing’s disease is the accumulation of large amounts of
fat. This suggests that glucocorticoids, at least in the intact animal, stimulate accumulation of lipid stores. When examined in vitro, pharmacological
levels (0.25–1.0 µM) of glucocorticoids induce differentiation of
preadipocytes, but only in the presence of insulin and/or serum. It is not
clear whether glucocorticoids are required for, or simply act synergistically
with other factors to accelerate adipocyte differentiation. Glucocorticoid
effects on adipogenesis are dependent upon the species and cell type, whether
primary cells or cell lines, studied. Glucocorticoids stimulate arachidonic
acid metabolism to produce the prostaglandin, prostacyclin (PGI2), and also
increase cellular cAMP concentrations. Both are factors that induce
adipocyte differentiation.
In mature adipocytes, glucocorticoids play a key role in the regulation of
carbohydrate and lipid metabolism. Dexamethasone modulates the expression of many genes involved in adipocyte energy metabolism, including the
repression of genes for the IRS-1, the β-adrenergic receptor and the glucose
transport protein. Dexamethasone induces the expression of genes for
adipsin, LPL, GDPH, phosphoenolpyruvate carboxykinase and pyruvate
kinase. These effects are mediated by the nuclear hormone glucocorticoid
receptor and which binds to GREs on specific target genes. In differentiated
3T3-L1 cells and adipose tissue, the effects of glucocorticoids are mediated
by the activation of C/EBPδ and the repression of C/EBPα transcription.
The effects of glucocorticoids act in synergy with C/EBPβ.
Prostaglandins and cAMP
Prostaglandins (PG) are lipid-derived, arachidonic acid derivatives produced by preadipocytes and adipocytes. The major prostaglandin in these
cells is prostacyclin (PGI2). Prostacyclin induces preadipocyte differentiation
and activates all PPAR (α, δ, γ) transcription factors. The release of prostacyclin by adipocytes from the OB 1771 cell line can induce differentiation of
preadipocytes. Prostacyclin is believed to be the factor that induces adult
adipose hyperplasia and differentiation in response to the maturation of fat
cells. Another prostaglandin, PGF2α inhibits adipocyte differentiation at
physiological concentrations (3×10−8 M). PGF2α also induces TGF-α expression in preadipocytes and adipocytes. Other prostaglandins, PGD2 and 15deoxy-PGJ2 may be natural PPARγ ligands, activating PPARγ and inducing
differentiation.
Hormones, Growth Factors and Adipose Tissue
177
cAMP, in general, enhances differentiation of preadipocytes. As discussed in the earlier section on preadipocyte differentiation in vitro, the
chemical IBMX inhibits phosphodiesterase-catalysed cAMP breakdown.
This elevates intracellular cAMP levels and induces preadipocyte differentiation. cAMP-mediated differentiation is accompanied by an induction of the
C/EBPβ and PPARγ transcription factors. Treatment of 3T3-L1 cells with
prostaglandins also induces cAMP accumulation. As with the effects of
prostaglandins on preadipocytes, the effects of cAMP on preadipocytes may
depend upon the stage of differentiation, inducing differentiation in early
stages and inhibiting it later.
Negative Regulators of Adipose Differentiation
There are several factors that act to oppose the stimulatory effects of hormones and growth factors on the differentiation of adipose tissue. EGF is a
6 kDa single-chain peptide initially discovered in mouse submaxillary salivary glands. It is also present in saliva, blood and urine. EGF promotes the
proliferation and differentiation of many mesenchymal and epithelial cell
types. EGF acts as a local growth factor in many tissues, including adipose
tissue. In preadipocytes, EGF stimulates proliferation and inhibits differentiation at very small, physiological doses (0.6 ng/ml is the 50% effective dose).
Treatment of newborn mice for 9 days with EGF decreases body weight, fat
mass and the proportion of mature adipocytes in fat depots, by delaying
adipocyte differentiation. TGF-α has a significant amino acid sequence
homology with EGF and acts via EGF receptor. It is produced by both
preadipocytes and adipocytes. Transgenic TGF-α mice display a 40–80%
decrease in fat pad weight with an overall decrease of 50% in total body fat.
Other potent mitogens such as FGF and PDGF increase preadipocyte proliferation and may inhibit differentiation, although the effects are variable and
species-dependent.
TGF-β is a potent inhibitor of adipocyte differentiation. The TGF-β
mRNA is present in proliferating preadipocytes and differentiated
adipocytes. TGF-β increases the synthesis of the ECM surrounding the
adipocytes, and this is believed to inhibit the differentiation of fat cells.
Retinoic acid, a vitamin A metabolite, is a potent inhibitor of adipocyte
differentiation in preadipocyte cell lines and primary cultures. All transretinoic acid binds to the RAR nuclear hormone receptor, which forms
dimers with the RXR receptor, the receptor for 9-cis-retinoic acid. Retinoic
acid and PPARγ competition for limited RXR may inhibit the stimulatory
effects of PPARγ on differentiation (recall that PPARγ forms dimers with
RXR). Alternatively, the RAR/RXR complex acts as a retinoic aciddependent activator or repressor of gene transcription. Retinoic acid acts
very early in the adipocyte differentiation programme, immediately after the
hormonal induction of differentiation, but before the expression of C/EBPα.
Retinoic acid acts to inhibit the expression of C/EBPα and PPARγ, but has no
effect on C/EBPβ expression.
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Chapter 8
Adipose tissue as an endocrine organ
Adipose tissue is not simply a static reservoir for lipid deposition and mobilization. It has recently been shown that adipocytes secrete a variety of local
and systemic factors which regulate appetite, energy homeostasis, the
immune response and vascular development. The newly discovered
endocrine properties of adipose tissue expand the functional role of this
seemingly innocuous tissue to that of a regulatory entity. New information
about the regulatory role of adipose tissue, at both the local and systemic levels, has led to a reassessment of the function of this tissue and the factors that
regulate metabolism and growth. In the future, this new functional role of
adipose tissue may lead to new methods for the regulation of animal production. Some of the factors secreted by adipocytes and their regulatory
functions are discussed in this section.
Leptin will be discussed fully in Chapter 11. Leptin is a prominent
secreted hormone of adipose tissue that acts in a classical endocrine manner.
It was the first adipose tissue hormone identified when it was discovered in
1994. Initially, there was a great deal of anticipation that this adipocytederived, appetite-suppressing, fat-reducing hormone would provide an
effective treatment for human obesity. Unfortunately, this has not proven to
be the case. Nevertheless, leptin remains an important hormone that regulates appetite, energy storage and fertility. A more complete understanding
of its effects may yet provide another means to regulate appetite, carcass
composition and animal production efficiency. Manipulation of its actions
on the reproductive system has the potential to provide major alterations in
the reproductive efficiency of farm animals.
Adiponectin is the most abundant gene product of human adipose tissue.
It occurs at very high levels in plasma (5–30 µg/ml). Adiponectin is also called
adipocyte complement-related protein of 30 kDa (Acrp30) and AdipoQ.
Adiponectin shares homologies with the immune protein complement Clq and
is thought to play a role in the inhibition of inflammatory pathways.
Adiponectin also has metabolic effects on lipid and carbohydrate in liver, skeletal muscle and adipose tissue. Adiponectin enhances insulin sensitivity in the
liver, reducing hepatic glucose output in response to insulin. The secretion of
adiponectin is induced by IGF-I, insulin and PPARγ ligands, such as the TZDs.
Its expression is suppressed by treatment with β-agonists and glucocorticoids.
TNF-α is a 17 kDa polypeptide that is named for its ability to induce
tumour necrosis when injected into tumours in vivo. Many tissues produce
TNF-α, including skeletal muscle, lymphoid tissue and the adipocytes in
adipose tissue. While TNF-α is found in the circulation, it likely acts locally
in adipose tissue by autocrine and paracrine mechanisms. This growth factor inhibits differentiation of preadipocytes into adipocytes in vitro. On the
other hand, TNF-α can induce dedifferentiation of mature adipocytes, acting
by a reduction of PPARγ and C/EBPα expression. TNF-α induces
preadipocyte and adipocyte apoptosis and induces lipolysis of fat stores
in vivo and in vitro. These observations suggest that TNF-α may function to
limit adipose tissue mass in the animal.
Hormones, Growth Factors and Adipose Tissue
179
Resistin is a 10 kDa polypeptide that is produced solely by adipose tissue. Its name is derived from early observations that resistin conferred
insulin resistance on obese rodents. It has since been shown that resistin is
not related to human insulin resistance. Resistin expression is increased during differentiation, by feeding, obesity and insulin. Resistin acts by
endocrine, autocrine and paracrine pathways to inhibit adipocyte differentiation, possibly via a feedback mechanism that restricts adipocyte formation.
Resistin expression is suppressed by fasting, diabetes and PPARγ agonists,
such as the TZDs. Its physiological role in the adult is still unknown.
The renin–angiotensin system is an osmoregulatory system that modulates fluid balance and vascular tone, playing an important role in the regulation of blood pressure. The components of the classical system are secreted
by the kidneys and liver and are found in the circulation. Renin, secreted by
the kidneys, is a proteolytic enzyme that cleaves hepatic-derived
angiotensinogen to angiotensin I, which is converted to the active octapeptide, angiotensin II. Adipose tissue also synthesizes all components of this
system and it is believed that the adipose-derived angiotensin II acts locally
by autocrine and paracrine mechanisms. Adipose tissue also contains receptors for angiotensin II. Local angiotensin II regulates adipose tissue blood
supply by stimulating angiogenesis during adipogenesis. Angiotensin II also
stimulates preadipocyte differentiation into adipocytes. This is mediated by
the stimulation of PGI2 synthesis. Angiotensin II stimulates lipogenesis in
mature adipocytes. Adipose tissue angiotensinogen production is upregulated by nutrients. This observation, as well as the effects of angiotensin II on
lipid metabolism, suggests that angiotensin may play a role in the regulation
of body weight and body composition.
Further Reading
Ailhaud, G., Grimaldi, P. and Negrel, R.
(1992) Cellular and molecular aspects of
adipose tissue development. Annual
Review of Nutrition 12, 207–233.
Butterwith, S.C. (1994) Molecular events in
adipocyte development. Pharmacology
and Therapeutics 61, 399–411.
Ferre, P. (2004) The biology of peroxisome
proliferator-activated receptors; relationship with lipid metabolism and insulin
sensitivity. Diabetes 53(Suppl. 1), S43–S50.
Gregoire, F.M. (2001) Adipocyte differentiation: from fibroblast to endocrine cell.
Experimental Biology and Medicine 226,
997–1002.
Gregoire, F.M., Smas, C.M. and Sul, H.S. (1998)
Understanding adipocyte differentiation.
Physiological Reviews 78, 783–809.
Hausman, G.J. (1989) The influence of
insulin, triiodothyronine (T3) and
insulin-like growth factor-I (IGF-I) on the
differentiation of preadipocytes in
serum-free cultures of pig stromal-vascular cells. Journal of Animal Science 67,
3136–3143.
Hausman, G.J., Jewell, D.E. and Hentges, E.J.
(1989) Endocrine regulation of adipogenesis. In: Campion, D.R., Hausman, G.J.
and Martin, R.J. (eds) Animal Growth
Regulation. Plenum Press, New York,
pp. 49–68.
Kim, S. and Moustaid-Moussa, N.
(2000) Secretory, endocrine and
autocrine/paracrine function of the
adipocyte. Journal of Nutrition 130,
3110S–3115S.
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Lazar, M.A. (1999) PPARγ in adipocyte differentiation. Journal of Animal Science
77(Suppl. 3), 16–22.
Louveau, I. and Gondret, F. (2004) Regulation
of development and metabolism of adipose tissue by growth hormone and the
insulin-like growth factor system.
Domestic Animal Endocrinology 27,
241–255.
Mondrup, S. and Lane, M.D. (1997)
Regulating adipogenesis. The Journal of
Biological Chemistry 272, 5367–5370.
Nawrocki, A.R. and Scherer, P.E. (2004) The
delicate balance between fat and muscle:
adipokines in metabolic disease and
musculoskeletal inflammation. Current
Opinion in Pharmacology 4, 281–289.
Ntambi, J.M. and Kim, Y.-C. (2000) Adipocyte
differentiation and gene expression.
Journal of Nutrition 130, 3122S–3126S.
Picard, F., Kurtev, M., Chung, N., ToparkNgarm, A., Senawong, T., de Oliveira,
R.M., Leid, M., McBurney, M.W. and
Guarente, L. (2004) Sirt1 promotes fat
mobilization in white adipocytes by
repressing PPAR-γ. Nature 429, 771–776.
Prins, J.B. (2002) Adipose tissue as an
endocrine organ. Best Practice and
Research. Clinical Endocrinology and
Metabolism 16, 639–651.
Rebuffe-Scrive, M., Krotkiewski, M.,
Elfverson, J. and Bjorntorp, P. (1988)
Muscle and adipose tissue morphology
and metabolism in Cushing’s syndrome.
Journal of Clinical Endocrinology and
Metabolism 67, 1122–1128.
Rosen, E.D., Walkey, C.J., Puigserver, P. and
Spiegelman, B.M. (2004) Transcriptional
regulation of adipogenesis. Genes and
Development 14, 1239–1307.
Serrero, G. and Lepak, N. (1996) Endocrine
and paracrine negative regulators of adipose differentiation. International Journal
of Obesity 20(Suppl. 3), S58–S64.
9
Steroids and Animal Growth
Steroids are complex, water-insoluble, lipid-like compounds which are synthesized from cholesterol. Ruminants are unique in that they are the only
animals which have a positive growth response to oestrogens, the female
steroid hormones. Oestrogens were the first growth promotants to be studied and used to enhance production efficiency in farm animals. In ruminants, oestrogens increase muscle growth and fat deposition of castrated
males. As they are relatively abundant, cheap and provide reliable responses
to enhance growth and alter body composition, they are still widely used in
animal production.
Types, sources and functions of the major naturally occurring steroids
are shown in Table 9.1. Steroids can be divided into two broad categories,
based upon their source and function: the first are the so-called sex steroids,
produced by the gonads (ovaries, testes) and the placenta, and, second, the
adrenal steroids. Glucocorticoids, also called corticosteroids and corticoids,
such as cortisol and corticosterone, are products of the adrenal cortex, the
outer portion of the adrenal gland. Physiologically, the adrenocortical
steroids are involved primarily in the mobilization of carbohydrates and
amino acids in response to food deprivation or stress. The function of these
hormones has been discussed in previous chapters. The focus of the current
chapter is on the sex steroids and their effects on farm animal growth.
The naturally occurring female sex steroids, produced by the ovaries,
include oestrogens, the most potent of which is oestradiol-17β. Oestrogens
function to regulate reproduction and are responsible for the secondary sexual characteristics of females, such as maintenance of the uterus and mammary glands, distribution and quantity of fat and muscle, and, in humans,
smooth skin, small larynx and lack of facial hair. Diethylstilbestrol (DES) is
a potent synthetic, non-steroidal oestrogen first synthesized in 1938. In the
late 1940s, it was shown to increase ADG in heifers, sheep and poultry. It was
patented in the early 1950s by Wise Burroughs as stilbestrol and was the
impetus for the formation of Elanco by the Eli Lily Corporation in 1954. DES
is no longer in use. It was banned in 1979, as a potential carcinogen. Other
oestrogens which are still widely used in ruminant animal growth stimulation
include oestradiol-17β, oestradiol benzoate, zeranol, dienestrol and
©K.L. Hossner 2005. Hormonal Regulation of Farm Animal Growth (K.L. Hossner)
181
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Chapter 9
Table 9.1. General characteristics of steroids.
Steroid type
Examples
Source
Function
Oestrogen
Oestradiol-17β
Ovary
Reproduction,
2° sexual characteristics
Progestins
Progesterone
Ovary, placenta
Reproduction,
2° sexual characteristics
Androgens
Testosterone
Testis
Reproduction,
2° sexual characteristics
Corticosteroids
(glucocorticoids)
Cortisol, corticosterone
Adrenal cortex
Carbohydrate
metabolism
Hexestrol®. The structures for oestrogens, progestins and androgens are
shown in Fig. 9.1.
Progestins are the second major family of naturally occurring sex steroids
in females. Progesterone is the naturally occurring progestin in females.
Progesterone has direct effects on the uterus, priming it for pregnancy by stimulating its growth and differentiation. Progesterone is also responsible for the
maintenance of pregnancy in mammals. Naturally occurring progesterone is
secreted by the corpus luteum of the ovary after ovulation and is elevated during the latter part of the oestrous cycle. If conception and pregnancy occur,
progesterone levels remain elevated, and along with oestrogen, progesterone
prepares the uterus to receive the embryo and maintain pregnancy. Removal
of progesterone results in the immediate termination of pregnancy.
Progestins, such as the naturally occurring progesterone and potent synthetics like melengestrol acetate (MGA), enhance growth and feed efficiency
Fig. 9.1. Chemical structures of oestrogens, androgens and progestins.
Steroids and Animal Growth
183
(FE) in cyclic heifers. This is thought to be primarily an effect on behaviour,
although there is some evidence that progesterone increases the half-life of
oestrogens when given in combination. The natural role of progesterone
involves not only effects on the uterus, but also includes behavioural effects
on the mother, inducing a relatively calm state, which is conducive to pregnancy. Oestrogens have the opposite effect, inducing erratic, frenzied behaviour in females during the heat phase of the oestrous cycle. The word
‘oestrus’ is derived from the Greek for ‘gadfly’, a term suggesting the erratic
behaviour seen in animals in heat. The positive effects of progestins in
enhancing farm animal growth are thought to be a result of the ‘calming’
effect of progestins in female animals. This counteracts the hyperactivity
induced by oestrogen and allows energy to be directed to body growth as
opposed to energy use in oestrogen-induced hyperactivity.
The androgen steroids are produced by the testis of the male and are the
predominant steroids in the male. Testosterone is the primary circulating natural androgen produced by the testes. Testosterone is converted to its active
metabolite, dihydrotestosterone (DHT), in most target tissues by the enzyme
5α-reductase. Androgens are responsible for the typical male secondary sexual characteristics, such as maintenance of the accessory sex glands (prostate,
seminal vesicles, bulbourethral gland) that produce seminal fluids, the external male genitalia and sperm production by the testis. They also have distinct
effects on the CNS. These effects are manifested in the induction of the sex
drive, or libido, in both sexes, as well as aggressive nature of male animals to
maintain dominance during the mating season. In humans, androgens induce
laryngeal growth, beard growth and hair loss. Aside from the reproductive
system, one of the primary target tissues for androgens is skeletal muscle. The
anabolic effects of androgens on male skeletal muscle are responsible for the
typical sexual dimorphism of mammals. In most species, males are larger,
with a greater proportion of muscle than females.
Another important function of the sex steroids includes effects on bone
growth and maturation. In the pre-pubertal animal, low doses of the sex
steroids increase linear bone growth and bone mass. The growth spurt at
puberty is largely the result of maturation of the gonads and the outpouring
of androgens in males and oestrogens in females. Later on, when growth
ceases, higher circulating levels of oestrogens and androgens stimulate the
epiphyseal growth plates to undergo fusion or closure as they transform
from cartilaginous structures into mature bone.
Several commercial formulations of anabolic steroids have been
approved by the Food and Drug Administration (FDA) as livestock production enhancers in the USA. The trade names, formulations and their target
animals are provided in Table 9.2.
Oestrogens and Ruminant Growth
It has long been known that castrated males have more fat tissue and less
lean tissue than non-castrated males. Thus, these animals have a lower FE
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Chapter 9
Table 9.2. Anabolic steroid formulations and their uses in farm animals.
Trade name
Composition
For use in
Synovex-S
20 mg Oestradiol benzoate
and 200 mg progesterone
Steers
Synovex-H
20 mg Oestradiol benzoate
and 200 mg testosterone
propionate
Heifers
Ralgro
36 mg Zeranol
Steers
Heifers
Calves
12 mg Zeranol
Lambs
MGA
Melengestrol acetate
0.25–0.50 mg/day
Heifers
Compudose
24 mg Oestradiol-17β
Steers
Heifers
Calves
Steer-oid
20 mg Oestradiol benzoate
and 200 mg progesterone
Steers
Synovex-C
10 mg Oestradiol-17β and 100 mg
progesterone
Calves
Heifer-oid
20 mg Oestradiol benzoate
and 200 mg testosterone
propionate
Heifers
Finaplix-S
140 mg Trenbolone acetate
Steers
Finaplix-H
200 mg Trenbolone acetate
Heifers
Revalor-S
120 mg Trenbolone acetate
and 24 mg oestradiol-17β
Steers
Revalor-H
140 mg Trenbolone acetate
and 14 mg oestradiol-17β
Heifers
than intact males. Oestrogen treatment of castrated male ruminants such as
wethers and steers increases ADG and FE by 10% to 20%, with only a small
increase in feed intake (FI). Along with these production effects there is an
increase in lean tissue deposition (including bone and connective tissue),
bone density and protein and water deposition. At the same time, subcutaneous and intramuscular fat, along with carcass grade, are reduced. Typical
effects of oestrogens on performance and carcass characteristics of steers are
Steroids and Animal Growth
185
shown in Table 9.3. These are the results after 164 days of treatment with
oestrogen implants. Steers were implanted with Synovex-S or Ralgro on day
1 only (single) or on day 1 and day 70 (reimplanted group). Oestradiol-17β
(E2) was given as a controlled-release silicon rubber implant. No statistical
differences were seen between treatments with oestradiol and progesterone
or Zeranol or oestradiol-17β. There were no differences in the source of
oestrogen on performance characteristics but characteristics were improved
by oestrogen treatment over non-treated animals.
When oestrogens are given to intact (non-castrated) ruminant males,
only small, variable effects are observed. There is a reduction of aggression
in bulls and, in general, there is an increase in body fat in both sheep and cattle. As might be expected, females have a much lower response to exogenous
oestrogens, which induce only minor effects.
Oestrogens and Non-ruminants
Oestrogens are less effective as production enhancers in non-ruminants,
although they do have effects on production and body composition. For
example, in boars, oestrogens increase ADG and per cent body fat, while
reducing boar odour (taint) in meat. In addition, oestrogens slightly increase
FI, FE and nitrogen retention in boars. In barrows and gilts, there are few if
any beneficial effects of oestrogen treatment. In these animals, oestrogen
increases udder growth with a concomitant reduction in belly (bacon) value.
In poultry, oestrogen treatment increases both subcutaneous and intramuscular body fat by 55%, while FI in increased by about 20%. This results in a
reduction of FE by 8%. For these reasons, oestrogens are not used as production enhancers in poultry.
Table 9.3. Effects of oestrogen implants on growth and carcass characteristics of steers.
Treatment
ADG (kg)
%
FI
(kg/day)
%
FE
%
Cutability
Control
1.22
–
8.32
–
6.91
–
49.1
Single
1.30
7.1
8.68
4.3
6.74
2.5
49.1
Reimplant
1.40
15.1
8.86
6.5
6.37
7.8
49.1
E2
1.38
13.2
8.73
4.9
6.39
7.5
49.0
From Basson et al. (1985). FE is given as feed consumed per unit gain. Improvement over control
animals is shown as %.
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Chapter 9
Androgens and Ruminant Growth
Androgens, the male reproductive hormones, also are used in regulating
farm animal growth. Unlike oestrogens, it is not particularly surprising that
androgens might be used as growth enhancers, as their normal physiological role in the male includes the stimulation of skeletal muscle growth. One
needs to look only at the larger body, heavy forequarters and relatively lean
form of a bull compared to steers or heifers to gain an appreciation for the
anabolic effects of androgens on body form and composition. Unfortunately,
androgens also have strong behavioural effects, inducing competition for
hierarchy and for dominance in mating. This includes, of course, typical
male behaviour such as aggressiveness and fighting. While an important
part of animal behaviour and successful propagation of the species, these
traits are unwanted in animal production systems. Like the gadfly female,
the bull-headed and combative male wastes energy (and induces bruises and
wounds) with this aggressive behaviour. Hence, farm animal males are traditionally castrated, in part to avoid these behaviours.
Androgens increase ADG and carcass quality in steers, lambs and
heifers. Trenbolone acetate (TBA) is a synthetic androgen that has strong
anabolic effects on growth, but is only weakly androgenic, eliminating many
of the unwanted behavioural traits. It was approved for use in steers and
heifers in 1987. Androgens are most effective in castrated males when they are
given in combination with oestrogen. This results in an additive response,
with improved production efficiency and carcass characteristics over that
seen with either steroid given alone (Table 9.4). Compared with oestrogen
treatment, TBA is less effective in inducing positive growth performance
characteristics, but has more dramatic effects on carcass composition. When
given in combination with oestrogen, additive effects are seen, with larger
increases in ADG and FE than seen with single steroid treatment.
As with oestrogens, the effects of androgens on animal growth and body
composition vary with the species and gender of the animal. In intact males,
Table 9.4. Effects of oestrogen and androgens given alone or in combination on growth and
carcass characteristics in steers.
ADG (kg)
%
FE
%
Yield grade
(YG)
Cutability
Control
1.39
–
6.79
–
2.51
49.96
E2
1.55
11.5
6.29
7.4
2.43
50.14
TBA
1.49
7.2
6.29
7.4
2.25
50.54
E2 + TBA
1.62
16.5
5.95
12.4
2.29
50.38
Treatment
Adapted from Trenkle (1987).
Steroids and Animal Growth
187
there is an increase in ADG and carcass leanness, with better FE than castrates. Androgen-treated males have larger forequarters, neck and crest than
females or castrated males, as these muscles are more androgen-sensitive. In
addition, behaviour problems typical of males, such as fighting, are common
in androgenized animals. In heifers, TBA increases ADG, nitrogen retention
and protein deposition. In beef and dairy cows, TBA increases carcass lean
tissue while reducing fat deposition.
Mechanism of Action of Steroids in Ruminant Growth
As noted above, ruminants are unique in their growth response to oestrogens. Oestrogens are female hormones that are primarily involved in the regulation of reproductive events. Compared to males, females are generally
smaller-framed animals, with smaller muscle masses and larger proportions
of body fat. Human females are characterized by their narrower shoulders,
wider hips, breasts and absence of facial hair. Thus, it is a bit of a paradox
that oestrogenic hormones would induce ruminant males to grow faster and
accumulate lean tissue mass. A lingering mystery in animal biology is ‘How
do oestrogens induce muscle growth in ruminants?’ Several hypotheses
have been introduced and examined experimentally.
Of course, the possibility that oestrogens have direct effects on growth is
the first that one would propose and examine. If oestrogens are having a
direct effect on skeletal muscle, they must act through muscle receptors and
one would expect to see either increased numbers of skeletal muscle oestrogen receptors or an increase in the response of ruminant skeletal muscle to
the direct effects of oestrogens. Oestrogen receptors are indeed present in
ovine and bovine skeletal muscle, but their concentrations are very low, i.e.
only 1% of uterine concentrations. In addition, similar levels of the oestrogen
receptors are also seen in rat muscle, which does not have a growth response
to oestrogen. These observations argue against a direct effect of oestrogens
on ruminant skeletal muscle.
Oestrogens may act by indirect mechanisms in the living animal.
Oestrogens may act by inducing another hormone, which, in turn, affects the
growth of muscle. With the knowledge that GH has significant and strong
effects on skeletal muscle growth and development, scientists sought evidence that perhaps circulating GH mediated the effects of oestrogens. This
hypothesis was supported by the observations that oestradiol-17β and GH
have similar effects on nitrogen balance in ruminants. Both act to conserve
body nitrogen stores and increase lean tissue deposition. This is reflected by
reduced urinary nitrogen and circulating amino acid concentrations in
response to both GH and oestradiol-17β. Evidence was accumulated that GH
mediates the effects of oestrogen by demonstrating that oestrogen treatment
increases pituitary weight and GH release in animals. In addition, oestrogen
treatment of sheep anterior pituitary cells grown in vitro induced the release
of GH by the cells. Although similar studies using bovine anterior pituitary
cells failed to demonstrate this release immediately, pretreatment of the cells
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Chapter 9
with oestradiol-17β sensitized the cells to the action of GHRH that then
increased the GH release. This provided tantalizing evidence that perhaps
oestrogens were acting via the pituitary/hypothalamic system to stimulate
GH release.
Unfortunately, there are problems with this hypothesis. For example, the
treatment of animals with both GH and oestradiol-17β results in additive
effects. This suggests that these compounds act through independent pathways. Both GH and oestradiol-17β stimulate nitrogen retention in ruminants, as reflected by a reduction of circulating urea nitrogen, amino acids
and urinary nitrogen. Oestrogens have effects similar to GH on production
characteristics such as ADG and FE, and carcass protein and water as well as
bone deposition. In addition, it has been shown that the positive effects of
oestrogens on protein deposition may result from an inhibition of protein
degradation. GH, on the other hand, has no effect on protein degradation
and increases muscle protein accretion by increasing protein synthesis. In
laboratory rodents that do not have a growth response to oestrogen, oestrogen also induces an increase in GH. These observations provide evidence
that the effects of oestrogen are not mediated by GH, at least in a straightforward manner.
Similarly, an indirect mechanism of oestrogen involving GH might act
through the GH receptor. Although GH receptors are present in many tissues, hepatic GH receptors are abundant and mediate the GH induction of
IGF-I secretion by the liver. IGF-I, in turn, mediates many of the effects of
GH, especially those in skeletal muscle. In steers implanted with oestrogen,
liver GH receptors are increased by 250%. This suggests that oestrogen
effects may be mediated by an increased hepatic sensitivity to GH, with no
or minor increases in circulating GH, resulting in increased circulating IGF-I.
Oestrogen implants increase plasma IGF-I levels, and this may provide an
explanation for the additive effects of oestrogen and GH, as GH also
increases IGF-I concentration in the circulation.
Androgen mechanisms of action
Interestingly, natural and synthetic androgens have different mechanisms of
action. Testosterone and DHT increase both muscle protein synthesis and
degradation, although the effect on stimulating protein synthesis predominates, resulting in net protein deposition. The synthetic androgen, TBA, on
the other hand, reduces both protein degradation and synthesis. As TBA has
a greater effect on decreasing protein degradation, there is an overall
increase in muscle protein deposition.
Like oestrogens, the mechanism of action of androgens in promoting
muscle growth and altering body composition is controversial. Skeletal muscle contains androgen receptors, which bind both testosterone and the TBA
metabolite, 17α-methyl-TBA. This provides a pathway for the direct action
of androgens on skeletal muscle growth.
Steroids and Animal Growth
189
In addition, several candidates which may mediate the effects of androgens have been investigated. It has been proposed that these indirect effects
of androgens are, in part, due to suppression of the effects of adrenal glucocorticoids. Corticosteroids reduce protein synthesis and stimulate protein
degradation, mobilizing amino acids for gluconeogenesis under conditions
of stress or food restriction. Treatment with TBA reduces cortisol levels while
increasing protein deposition. This suggests that cortisol may play a permissive role in mediating androgen effects and that reduction of endogenous
corticosteroid levels by androgens allows the anabolic effects of androgens
to be expressed.
Likewise, circulating concentrations of the thyroid hormones, specifically T4, are reduced by androgen treatment. The natural role of T4 is to
increase energy expenditure in the form of increased oxidative metabolism
and muscle protein degradation. Thus, the reduction of T4 concentrations by
androgens may contribute to the enhanced energy efficiency of treated animals, as energy requirements and protein degradation are reduced in the
treated animal.
Finally, the effects of androgens on ruminant growth may actually be
mediated by oestrogens. Androgens are converted by peripheral tissues,
including skeletal muscle, into oestrogens, which may act as a local stimulant for muscle growth. Treatment of animals with TBA implants decreases
the clearance rate of oestrogens in steers, heifers and ewes and increases
concentrations of circulating oestradiol-17β. All of these processes lead to
increased concentrations of oestradiol-17β in the circulation, and to the
speculation that, in addition to direct effects of androgens mediated by
skeletal muscle receptors, some effects of androgens may be mediated by
oestrogens.
Treatment of ruminants with a combination of oestrogens and androgens leads to additive effects in steers, providing optimal growth rates in
castrated animals. This combination treatment has no effect on GH concentrations of treated animals, suggesting that effects of combination treatment may be direct, without involvement of the endocrine somatotrophic
axis.
In light of our relatively recent knowledge that some hormones, such as
IGF-I, can act as both endocrine factors and local growth factors, without the
intervention of the circulatory system, re-examination of the interactions of
steroids, GH and IGF-I at the local level have provided new insight into the
regulation of animal growth by anabolic steroids. In steers treated with
Revalor-S (TBA + oestradiol-17β) for 32 to 38 days, IGF-I mRNA levels in
liver and semi-membranous muscle were increased by 50% to 70%, while
circulating IGF-I was increased by 30% to 40%. In addition, this treatment
induced the number of actively proliferating satellite cells isolated from the
semi-membranous muscle. This suggests that a combination of oestrogenic
and androgenic steroids induced synthesis of IGF-I in skeletal muscle, and
the increased local IGF-I activated the normally resting satellite cells in a
local autocrine or paracrine manner (White et al., 2003).
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Chapter 9
References and Further Reading
Basson, R.P., Dinusson, W.E., Embry, L.,
Feller, D.L., Gorham, P.E., Htiryrt, H.P.,
Hinman, D.D., McAskill, J., Parrott, C.,
Riley, J., Stanton, T.L., Young, D.C. and
Wagner, J.F. (1985) Comparison of the
performance of estradiol silicone rubber
implant-treated steers to that of zearalanol or estradiol + progesterone.
Journal of Animal Science 61, 1023–1036.
Burroughs, W., Culbertson, C.C., Kastelic, J.,
Cheng, E. and Hale, W.H. (1954) The
effects of trace amounts of diethylstilbestrol in rations of fattening steers. Science
120, 66–68.
Buttery, P.J. and Sinnett-Smith, P.A. (1984)
The mode of action of anabolic agents
with special reference to their effects on
protein metabolism – some speculations.
In: Roche, J.F. and O’Callagan, D. (eds)
Manipulation of Growth in Farm Animals.
Martinus Nijhoff, The Hague, The
Netherlands, pp. 210–232.
Duckett, S.K. and Andrae, J.G. (2001) Implant
strategies in an integrated beef production system. Journal of Animal Science
79(Suppl. E), E110–E117.
Hancock, D.L., Wagner, J.F. and Anderson,
D.B. (1991) Effects of estrogens and
androgens on animal growth. In:
Pearson, A.M. and Dutson, T.R. (eds)
Advances in Meat Science, Vol. 7. Elsevier
Applied Science, New York and London,
pp. 255–297.
O’Lamhna, M. and Roche, J.F. (1984) Recent
studies with anabolic agents in steers and
bulls. In: Roche, J.F. and O‘Callaghan, D.
(eds) Manipulation of Growth in Farm
Animals. Martinus Nijhoff, The Hague,
The Netherlands, pp. 100–111.
Roche, J.F. and Quirke, J.F. (1986) The effects
of steroid hormones and xenobiotics on
growth of farm animals. In: Buttrey, P.J.,
Haynes, N.B. and Lindsay, D.B. (eds)
Control and Manipulation of Animal
Growth.
Butterworths,
London,
pp. 39–51.
Trenkle, A. (1987) Combining TBA, estrogen
implants results in additive growth promoting effects in steers. Feedstuffs 26, 43.
White, M.E., Johnson, B.J., Hathaway, M.R.
and Dayton, W.R. (2003) Growth factor
messenger RNA levels in muscle and
liver of steroid-implanted and nonimplanted steers. Journal of Animal Science
81, 965–972.
10
Catecholamines, Beta-agonists
and Nutrient Repartitioning
Living organisms are subjected to stress on a daily basis. Animals living in
the wild must deal with environmental challenges, such as cold, heat, rain,
wind or snow, as well as predator attacks and competition for food, habitat
and reproductive partners. Many of these daily stresses have been minimized or eliminated in domestic animals that are provided with food, shelter and protection from predators. Nevertheless, the survival response to
daily challenges is an innate characteristic that allows an animal to cope with
the daily stresses to which it is subjected. This highly evolved physiological
system that ensures an animal will survive stress, prosper and propagate is
regulated by components of the CNS, the autonomic nervous system and the
endocrine system.
When an animal is challenged by a threat, such as a predator or a mating competitor, it can respond in two ways. It can either defend itself, its
mate and offspring, or it can flee for safety, hoping to elude a more powerful foe. This behavioural response to stress was termed the ‘fight or flight’
syndrome by W.B. Cannon in 1932. This self-preservation response is accompanied by an array of acute physiological adaptations that mobilize energy
and provide it to organ systems involved in this reflex. The sympathetic portion of the autonomic nervous system and the adrenal glands are largely
responsible for changes in the cardiovascular, respiratory and gastrointestinal systems that mediate the response to stress. These processes are stimulated by the release of catecholamines such as norepinephrine, a
neurotransmitter in the SNS, and the adrenal gland hormone epinephrine.
This results in the mobilization of lipids and glycogen and their catabolism
by lipolysis and glycogenolysis to provide energy to the animal. In addition,
coordinated responses in the cardiovascular system occur. The rate and
strength of heart contractions are increased and vasoconstriction of blood
vessels reduces blood flow to the gastrointestinal tract. Concomitantly,
vasodilation of blood vessels increases blood flow to skeletal muscle, the
heart and the brain. In total, these physiological adaptations ensure that
organs involved in response to ‘fight or flight’ stress receive the energy
needed for optimal function. While one might properly assume that a
response to acute stress that involves the mobilization and use of body
©K.L. Hossner 2005. Hormonal Regulation of Farm Animal Growth (K.L. Hossner)
191
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Chapter 10
energy stores is a process that is counter to the use of energy for growth,
nevertheless, it has been recently found that catecholamine derivatives are
useful as agents to alter food animal body composition and improve production efficiency.
The Autonomic Nervous System and the Adrenal Medulla
The autonomic nervous system is part of the peripheral nervous system that
lies outside of, but arises from the CNS. The autonomic nervous system is
responsible for the innervation of the skin, visceral organs, blood vessels,
smooth muscles and glands of the body and regulates the involuntary reactions that characterize the functions of these tissues. The autonomic nervous
system is divided into two portions, the SNS and the parasympathetic nervous system (Fig. 10.1). Parasympathetic neurones originate in the craniosacral regions of the spinal cord, while those of the SNS arise from the
Fig. 10.1. Overview of the sympathetic and parasympathetic nervous systems.
Catecholamines, Beta-agonists and Nutrient Repartitioning
193
thoracolumbar regions of the spinal cord. In contrast to the somatic nervous
system, in which a single continuous nerve fibre extends to innervate muscle, neurones from the autonomic nervous system form synapses once they
have left the CNS. After leaving the spinal cord, the preganglionic neurones
for the autonomic nervous system terminate in ganglia, clusters of neuronal
cell bodies where the synapses occur (Fig. 10.2). The nerve fibres then continue on to their target tissues as postganglionic neurones. Sympathetic ganglia lie close to the spinal cord in a distinct chain of neurones, the
sympathetic trunk, or they are located halfway between the spinal cord and
the affected organ. In contrast, parasympathetic ganglia are located within
the walls of the affected organ, characterized by a short postganglionic fibre.
In both pre- and postganglionic parasympathetic neurones acetylcholine
acts as the neurotransmitter. On the other hand, the SNS uses acetylcholine
as the preganglionic neurotransmitter but the postganglionic neurotransmitter is norepinephrine, a catecholamine.
The adrenal medulla is a specialized modification of the SNS and is part
of the sympathoadrenal system. This system includes the SNS and the catecholamine hormone secretions of the adrenal medulla. Like the pituitary
gland, the adrenal gland is derived from more than one embryonic source.
The outer cortex, source of the glucocorticoids, is of mesodermal origin. The
inner medulla, which synthesizes and secretes the catecholamines, is
derived from embryonic neural crest cells, which arise near the spinal cord.
Central nervous system
Peripheral nervous system
Effector organ
Somatic nervous system
Acetylcholine Skeletal
muscle
Ganglion
Preganglionic axon
Postganglionic axon
Acetylcholine
Sympathetic
Norepinephrine
Adrenal medulla
Smooth and
cardiac muscle,
glands
Epinephrine
Autonomic
nervous
system
Acetylcholine
Ganglion
Preganglionic axon
Postganglionic axon
Parasympathetic
Acetylcholine Acetylcholine
Fig. 10.2. Comparison of the somatic and autonomic nervous systems, their ganglia and
neurotransmitters.
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Chapter 10
The preganglionic neurones that regulate the adrenal medulla arise from the
spinal cord and terminate in the adrenal medulla. Unlike other sympathetic
nerves, however, there is no postganglionic nerve fibre and the adrenal
medulla, instead, secretes its products, the catecholamine hormones, directly
into the bloodstream. The primary catecholamine produced and secreted by
the adrenal medulla of most mammals is epinephrine, a hormone closely
related to norepinephrine, the neurotransmitter of postganglionic sympathetic nerves. Epinephrine is released in response to preganglionic nerve
stimulation from the SNS and influences adrenergic receptors throughout
the body.
In mammals, the most abundant catecholamines are adrenaline (also
called epinephrine), noradrenaline (norepinephrine) and dopamine. The catecholamines are amino acid derivatives that are synthesized from tyrosine
(Fig. 10.3). This involves an initial hydroxylation of tyrosine to form dihydroxyphenylalanine (DOPA), followed by a decarboxylation resulting in
dopamine formation. Hydroxylation of dopamine produces norepinephrine,
which is then methylated to form epinephrine. The presence of the amino
methyl group on epinephrine distinguishes it from norepinephrine (the prefix ‘nor’, means ‘without’). As shown in Table 10.1, epinephrine, norepinephrine and dopamine are found in the circulation in very low levels which
are quite variable between species. Although norepinephrine and dopamine
are found in the circulation, they are primarily neurotransmitters that function within the nervous system. Very high concentrations of norepinephrine
are needed to induce a response in endocrine target organs. For example, in
humans, norepinephrine concentrations above 1800 pg/ml are needed to
Fig. 10.3. Biological synthesis of catecholamines.
Catecholamines, Beta-agonists and Nutrient Repartitioning
195
Table 10.1. Plasma concentration (pg/ml) of catecholamines in various species.
Species
Catecholamine
Epinephrine
Norepinephrine
Dopamine
Cattle
Rats
Humans
Cats
56
175
64
73
152
509
203
603
91
84
84
276
Source: Buhler et al. (1978).
stimulate metabolic and cardiovascular events. Dopamine acts primarily
within the brain, via specific dopamine receptors and is not considered a
hormone. Circulating concentrations of norepinephrine and dopamine are,
in most mammals, considered to result from ‘spillover’ into the blood during sympathetic activation.
Catecholamines function as the primary mediators of the response to
real or perceived stress from internal or external sources. The direct connection of the adrenal medulla to the CNS means that these compounds are rapidly released in response to external stressors. The release of catecholamines
from the adrenal medulla serves to integrate the CNS and SNS with the
endocrine system. The pathway involved in the stimulation of release of the
catecholamines from the adrenal medulla is shown in Fig. 10.4.
In response to events that reduce blood glucose, such as sudden stress,
fasting and exercise, epinephrine acts to rapidly mobilize energy reserves by
increasing glycogenolysis and gluconeogenesis in the liver and glycogenolysis in skeletal muscle. This results in a rapid increase in blood glucose.
Epinephrine treatment also suppresses pancreatic insulin release and
induces glucagon secretion. These effects combine to assure that a high level
of blood glucose is maintained for use as an energy source. In addition, lipolysis of adipose tissue is enhanced, providing free fatty acids and glycerol
that can be metabolized for energy production or used to produce additional
glucose by the process of gluconeogenesis.
Adrenergic Receptors
Like all hormones, catecholamines act through specific receptors on their target tissues. The receptors for catecholamines are complex, seven transmembrane domain, G-protein-linked membrane receptors which exist in several
forms. The two fundamental types of adrenergic receptors are called α and
β receptors. These receptors are present in many tissues and their proportions relative to one another determine the tissue response to adrenergic
stimulation. α-Adrenergic receptors, in general, increase smooth muscle contraction in response to stimulation. The relative sensitivity of α receptors
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Stress
↓
Central nervous system
Cerebral cortex
Limbic system
↓
Preganglionic cholinergic neurones
↓
Acetylcholine
↓
Adrenal medulla
↓
Epinephrine
Fig. 10.4. Control of epinephrine release from the adrenal glands.
to adrenergic agents is epinephrine > norepinephrine > isoproterenol (a synthetic β-agonist). α-receptors are further subdivided into α1 and α2 subtypes. α1 receptors mediate vasoconstriction, acting through G-proteins and
the phosphoinositides IP3 and DAG, resulting in the release of calcium
within the cell. α2 receptors are present in the presynaptic terminal axon of
sympathetic neurones. These act as negative feedback receptors, which
inhibit synaptic norepinephrine release.
While the α-receptors mediate synaptic events within the SNS, vasoconstriction and smooth muscle contraction, the predominant receptors for
adrenergic agents are the β-adrenergic receptors. In general, these receptors
induce smooth muscle relaxation and β-adrenergic receptors are more
sensitive to epinephrine than norepinephrine. The β-adrenergic receptor isoforms are 64 kDa receptors, which are indistinguishable by immunological
methods, share about 50% amino acid homologies, but have distinct functions. Ligand binding to the β receptor activates the enzyme adenylate cyclase,
which catalyses the conversion of ATP to cAMP in the cytoplasm (Fig. 10.5).
cAMP binds to PKA, activating this enzyme. PKA, in turn, phosphorylates
and activates several cytoplasmic proteins, including hormone-sensitive
lipase, the rate-limiting enzyme that catalyses triacylglycerol degradation in
the adipocyte. In addition, activated PKA phosphorylates the cAMP
response element binding protein (CREBP). CREBP binds to the cAMP
Catecholamines, Beta-agonists and Nutrient Repartitioning
197
Epinephrine
β-Adrenergic
receptor
Plasma membrane
Adenylate
cyclase
Cytoplasm
(+)
ATP
cAMP + PPi
Protein kinase A
(inactive)
Protein kinase A
(active)
Phosphorylation
Activated CREBP Activated hormone- Inactivated acetyl-CoA
sensitive lipase
carboxylase
Nucleus
Triacylglycerols
CREBP
CRE
Reduced fatty acid
synthesis
Fatty acids +
glycerol
Gene
transcription
Fig. 10.5. Mechanism of action of catecholamines in adipose tissue.
response element in the regulatory portion of affected genes, where it activates gene transcription. PKA phosphorylation of other proteins results in
their inactivation. For example, ACC, the rate-limiting enzyme for fatty acid
synthesis, is inactivated by phosphorylation. Overall, these effects enhance
lipolysis while reducing lipogenesis.
The β-adrenergic receptors are subdivided into three subtypes: β1, β2
and β3 receptors. β1 receptors mediate lipolysis in adipose tissue and other
effects in the cardiac muscle and intestinal smooth muscle. They respond
equally to circulating epinephrine and neural norepinephrine but are considered to be the primary neural system adrenergic receptor. They are the
most abundant subtype of β-adrenergic receptor in most tissues, and
account for about 80% of adipose tissue β receptors, 70% of heart, 65% of
lung, 60% of skeletal muscle and 50% of liver.
β2 receptors are the major receptors for circulating epinephrine and
mediate bronchodilation, vasodilation, uterine smooth muscle relaxation
and glycogenolysis in the liver. They are also thought to be essential in mediating the response to β-agonists in skeletal muscle. In contrast to the β1
receptors, epinephrine is much more effective in stimulating β2 receptors
than norepinephrine, which interacts weakly with the β2 receptor.
β-3 adrenergic receptors are the least abundant of the receptor subtypes.
In humans and pigs, the β3 receptors constitute less than 10% of the
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β-adrenergic receptor subtypes in adipose tissue and are less than 2% of the
β-adrenergic receptors in other tissues. In rodents, however, the β3 receptor
is the predominant subtype present in white and brown adipose tissue. The
β3 receptor is believed to mediate thermogenesis and lipolysis in rodent adipose tissues. This complex system of five receptors results in a very finetuned system in which tissues with multiple adrenergic receptors respond in
accordance with the ratio of α to β receptors present in the specific tissue.
β-Adrenergic agonists
Several β-adrenergic agents have been synthesized which interact with
β receptors. Some synthetic chemicals bind to β-adrenergic receptors and do
not elicit a response, but block the effects of the receptor. These are termed
β-receptor antagonists. Other synthetic chemicals bind to specific β-adrenergic receptors and mimic the effects of catecholamines. These are the β-receptor agonists, or β-agonists for short. In human and veterinary medicine,
β-agonists are used as bronchodilators to treat asthma, to stimulate cardiac
contraction strength or rate and to induce uterine relaxation. β-Agonists are
active when given orally and can be used as feed supplements in animal production systems. The compounds used in farm animal production have
enhanced effects on skeletal muscle and white adipose tissue and, at the
doses used, have reduced effects on the cardiovascular system. β-Agonists
used in animal production systems are called ‘repartitioning agents’ due to
their effects on redirecting nutrients for use by skeletal muscle at the expense
of adipose tissue. β-Agonists are effective, to varying degrees, in several
species including chickens, swine, sheep and cattle. In general, they are more
effective in cattle and sheep than in swine and induce better responses in
swine than in poultry.
Examples of synthetic β-agonists are shown in Fig. 10.6. These include
isoproterenol, a potent β-agonist that interacts with both β1 and β2 receptors
better than the naturally occurring catecholamines. Clenbuterol and
cimaterol are β2-agonists. Clenbuterol is used to induce bronchodilation in
horses and reduce uterine contraction in pregnant cows, but is not used as a
repartitioning agent in food animals. Ractopamine is a synthetic β-adrenergic receptor agonist that interacts with both β1- and β2-adrenergic receptors.
Ractopamine was the first β-agonist to be approved for use in meat animals
as a production enhancer, when it was approved by the FDA for use in swine
in 2000, under Elanco’s trade name of Paylean®. In 2003 it was approved for
use in finishing cattle, under the trade name of Optaflexx®.
Effects of β-adrenergic agonists on growth and body composition
When used as feed supplements in farm animals, β-agonists increase rate of
weight gain, improve feed efficiency, reduce fatness and increase carcass
protein deposition of treated animals (Fig. 10.7). The effects of β-agonists on
Catecholamines, Beta-agonists and Nutrient Repartitioning
199
Fig. 10.6. Examples of β-agonists and their β-adrenergic receptor specificity.
animal production are species-specific and depend upon the β-agonist used.
These effects are thought to be due to direct actions upon the tissues of interest, skeletal muscle and adipose tissue. Treatment with β-agonists has no
effect on circulating hormones and β-adrenergic receptors in tissues affected
by β-agonists are believed to mediate the effects of β-agonists.
Species
Parameter
Sheep
Cattle
Pigs
Chicken
Feed
intake
+2
−5
−5
−
Feed
efficiency
+15
+15
+5
+2
Weight
gain
+15
+10
+4
+2
Muscle
+25
+10
+4
+2
Fat
−25
−30
−8
−7
Fig. 10.7. Summary of the effects of β-agonists on production characteristics in various farm
animals, as percentage changes from untreated animals. Adapted from Moloney et al. (1991).
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Chapter 10
β-Agonists are the most potent known inducers of skeletal muscle
hypertrophy. For example, treatment of lambs with clenbuterol for 2 months
increases overall muscle mass by 25% to 30%, with an increase of 40% seen
in the gastrocnemius muscle. This is primarily an effect on muscle hypertrophy, as there is no increase in the DNA content in affected skeletal muscles.
The muscle protein accretion in response to β-agonists is due primarily to a
reduction in protein degradation. A decrease in both lysosomal protease
activity (cathepsin B) and in non-lysosomal proteinase activity (e.g. calpain µ)
is observed with β-agonist treatment. Reduced calpain µ activity is due to an
increase in the calpain inhibitor, calpastatin. Muscle hypertrophy and protein accretion is rapid and is measurable within 2 days of treatment in rats,
and is specific for skeletal muscle. Cardiac and smooth muscle are unaffected. The effects on skeletal muscle are time-dependent, characterized by
rapid early growth, which is later attenuated. Attenuation may be due to
downregulation of β receptors in skeletal muscle. Studies in rodents show
that receptor concentration is reduced by 50% after 18 days of treatment with
clenbuterol. This attenuation can be avoided by intermittent treatment,
e.g. treating for 2 days and resting for 2 days.
About 40% of the hogs in the USA are fed the β-agonist ractopamine
(Paylean®). As with any production enhancer, animals must be provided
with adequate nutrition, especially amino acids balanced in relation to
lysine, to provide the increased demand for lean deposition. β-Agonists are
provided as feed supplements and are most effective when given to older,
heavier animals. Low doses, in parts per million, are most effective and
weight gain is reduced when higher doses – more than 20 mg/kg – are fed.
This is due to a reduction of appetite and lower feed intake at higher doses.
Problems with metabolic acidosis and slow, downer pigs can occur. Pigs
treated with ractopamine have been likened to weightlifters. They walk
slowly and stiffly and take longer to load into transportation vehicles. As
they are highly susceptible to stress, they must be treated gently, not
crowded and not herded with electric prods.
A significant drawback to the use of β-agonists in many farm animals is
the increased toughness, as measured by Warner–Bratzler shear force, of the
resulting meat products. This is especially prominent in treated lambs, in
which the toughness of meat is more than doubled compared to non-treated
animals. Chickens and cattle are less affected, but still show a 15% to 35%
decrease in muscle tenderness. The tenderness of meat of ractopamine-treated
pigs is unaffected by treatment. The effects of β-agonists are specific for muscle fibre type, consistently increasing the cross-sectional area of Type II (fast,
white, glycolytic) fibres, while the trophic effects of β-agonists on Type I fibres
(slow, red, oxidative) are inconsistent. When animals are treated for long periods with β-agonists, there is a switch of fibre types, from Type I to Type II. The
long-term implications of this alteration of fibre type are unknown. The effects
of β-agonists on skeletal muscle are thought to be mediated by the β2 receptor,
the predominant adrenergic receptor in skeletal muscle.
Catecholamines, Beta-agonists and Nutrient Repartitioning
201
References and Further Reading
Buhler, H.U., Da Prada, M., Haefely, W. and
Picotti, G.B. (1978) Plasma adrenaline,
noradrenaline and dopamine in man and
different animal species. Journal of
Physiology (London) 276, 311–326.
Etherton, T.D. and Smith, S.B. (1991)
Somatotropin and β-adrenergic agonists:
their efficacy and mechanisms of action.
Journal of Animal Science 69 (Suppl. 2), 2–26.
Hadley, M.E. (2000) Catecholamines and the
sympathoadrenal system. In: Endocrinology, 5th edn. Prentice-Hall, Upper
Saddle River, New Jersey, pp. 338–361.
Mersmann, H.J. (1998) Overview of the
effects of β-adrenergic receptor agonists
on animal growth including mechanisms
of action. Journal of Animal Science 76,
160–172.
Moloney, A., Allen, P., Joseph, R. and Tarrant,
V. (1991) Influence of beta-adrenergic
agonists and similar compounds on
growth. In: Pearson, A.M. and Dutson,
T.R. (eds) Growth Regulation in Farm
Animals, Vol. 7. Elsevier Applied Science,
New York, pp. 455–513.
Moody, D.E., Hancock, D.L. and Hancock,
D.B. (2000) Phenethanolamine repartitioning agents. In: D’Mello, J.P.F. (ed.)
Farm Animal Metabolism and Nutrition.
CAB International, Wallingford, UK,
pp. 65–96.
Stock, M.J. and Rothwell, N.J. (1986) Effects of
β-adrenergic agonists on metabolism and
body composition. In: Buttery, P.J.,
Haynes, N.B. and Lindsay, D.B. (eds)
Control and Manipulation of Animal Growth.
Butterworths, London, pp. 249–257.
Stoller, G.M., Zerby, H.N., Moeller, S.J., Baas,
T.J., Johnson, C. and Watkins, L.E. (2003)
The effect of feeding ractopamine
(Paylean) on muscle quality and sensory
characteristics in three diverse genetic
lines of swine. Journal of Animal Science
81, 1508–1516.
11
Leptin, Body Composition
and Appetite Control
Regulation of food intake, metabolism and body composition is of prime
importance to farm animal producers. Animals do not thrive without adequate feed intake, and those that do not efficiently utilize feed intake are not
economically competitive. Animals that are overly fat or lean are unprofitable for producers and unacceptable to consumers. Much of an animal
producer’s effort and resources are directed towards providing adequate
nutrition to his animals and small alterations in feed consumption and efficiency are important in providing an economical return on invested
resources. Thus, the control of feed intake, the subsequent utilization of
nutrients and the ultimate production of a desirable meat product are linked.
The regulation of appetite at the cellular, molecular and endocrine levels is
only now being understood and utilized by animal producers.
In a broad perspective, food preferences are largely innate (genetic) in
animals. Carnivores, of course, are not physiologically adapted to digest,
absorb and flourish on a high roughage vegetarian diet, while herbivores
have not evolved to survive on a high protein, meat-based diet.
Nevertheless, within bounds, food preferences are also a learned behaviour.
Animals can be taught the avoidance of certain foods, either as a result of
sickness from consuming a toxic substance or in the laboratory by behavioural modification. Likewise, food that produces pleasurable sensations to
the animal is readily consumed. Learned food consumption is especially evident in omnivores such as humans, who have a wide range of diet options,
from vegan to vegetarian to carnivorous. In developed countries, frequently
these preferences are a result of parental guidance or pressure from peers or
commercial sources and are thus learned, not innate. Geographical circumstances also play a large role in the selection and consumption of foods. As
an extreme, the diet of an Inuit, for example, consists primarily of protein
and fat, derived from fish and marine mammals, with essentially no vegetables. More temperate latitudes provide a more diversified diet.
Ingestion of food is a complex process, regulated by sensory, mechanical, chemical, hormonal and neural mechanisms. The consumption of food
is a result of an initial positive sensory evaluation (Fig. 11.1). This includes
the sight and smell of the food, followed by oral evaluation through mastication. This provides additional sensory inputs, including the food’s texture
202
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Leptin, Body Composition and Appetite Control
203
Fig. 11.1. Regulation of food ingestion.
and taste. The food can be rejected at this point. If it is not, the food is swallowed, digested and absorbed into the body by the gastrointestinal tract.
Lipids are absorbed into the lymphatic system, while protein and carbohydrate (or VFA in ruminants) nutrients are taken directly into the bloodstream. Most of these blood-borne nutrients are absorbed into the hepatic
portal system where they are transported to the liver for oxidation and
energy production or transformed into storage products.
The induction of feeding behaviour and feed intake and the cessation of
this process when the animal is full (satiety) maintains the animal’s energy
balance and ensures metabolic homeostasis. Energy intake, in the form of
nutrients, is correlated with body weight. Reduced food intake results in an
increase in energy expenditure as animals increase the oxidation of substrates via intermediary metabolism in an effort to maintain body weight.
Excess food energy is efficiently converted into fat stores. At the same time,
oxidation of energy reserves is reduced. These responses to excess energy
availability result from evolutionary adaptations that favour energy storage
over dissipation. This provides a survival advantage for animals in the wild.
These animals, unlike domesticated animals, undergo sporadic periods of
fasting when food is scarce or unavailable. During times of food scarcity,
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Chapter 11
animals derive their energy from the metabolism of fat stores that were accumulated during periods of food abundance. Survival mechanisms that
favour energy storage, weight gain and fat accumulation predominate,
while there are few mechanisms that favour the loss of body weight. Thus,
the development of fatness in animals and obesity in humans is due to the
evolutionary advantages of excess food consumption and storage.
It should be emphasized at the outset that the regulation of appetite,
food-seeking or food-aversive behaviour and food intake are all processes
that are ultimately regulated by the CNS, and specifically, the hypothalamus.
It has long been known that specific areas of the hypothalamus, the ARC
nucleus, the ventromedial hypothalamus (VMH) and the lateral hypothalamus (LH), regulate feeding and satiety. Destruction of the VMH leads to
hyperphagia and obesity, while lesions in the ARC or LH induce anorexia
and weight loss. These observations suggested that there were specific, distinct feeding and satiety centres in the hypothalamus, but more refined studies support a more diffuse integration of food intake and satiety. Inputs into
the CNS may be neural, nutrient-mediated or hormonal and the regulation
of appetite and thus energy homeostasis can be acute or chronic. The focus
of this chapter is on the hormonal control of long-term energy homeostasis
by the adipose hormone leptin, but we will first discuss the factors that have
traditionally been studied as short-term regulators of food intake.
Theories on the Short-term Regulation of Food Intake
Over the past half-century, many theories of the control of food intake have
been proposed. One of the earliest was the thermostatic theory of Brobeck
(1948), which proposed that animals eat to maintain a constant body temperature. This was shown to be true only in special circumstances, e.g. to
avoid hypothermia. In 1953, Mayer proposed the glucostat theory, which suggested that the concentrations of glucose in the bloodstream played a negative feedback role in feed intake. This was supported by the correlation of
satiety and elevated blood glucose concentrations after a meal. In addition,
glucose infusion into monogastric animals reduced feed intake, although
glucose infusion had no effect on ruminant feed intake. It is now known that
specific regions of the hypothalamus that control feeding are sensitive to
changes in glucose concentrations and glucose, either directly, or indirectly,
through insulin or leptin, plays a significant role in the acute regulation of
feed intake in monogastrics. In ruminants, the products of microbial carbohydrate metabolism, the VFAs, subsume the role of glucose.
Mechanical and chemical properties of food also play a role in the regulation of food intake. This is especially important in ruminants, which due to
the large reservoir of feed in the rumen are subject to the inhibitory effects of
gut-fill. In this case, the physical capacity of the rumen determines, to some
extent, the feeding behaviour and food intake of the animal. Animals with a
full rumen have reduced feed intake. As the rumen empties, feed intake
increases. This effect is not strictly physical, but also depends upon
Leptin, Body Composition and Appetite Control
205
the chemical and osmotic properties of the consumed food and thus likely
involves chemical sensing via chemoreceptors as well as the mechanoreceptors of the gut. The physical effects of gut fill are mediated by stretch receptors in the rumen, but the effects of feed composition suggest that nutritional
and hormonal mechanisms are also involved. This is, like the regulation by
blood glucose and insulin, a short-term regulatory mechanism.
The Long-term Regulation of Food Intake – the Lipostat Theory
The early theories discussed above on the short-term regulation of eating
were shaped by the scientific environment of the times. Thus, what may
seem to us as the somewhat obvious effects of ‘being full’, or body temperature and circulating glucose concentrations were based on factors that were
known at the time to be involved in regulating food intake. While all of these
theories are still valid to some extent, they apply primarily to the acute regulation of food ingestion. The lipostat theory was proposed by Kennedy in 1953
to explain the regulation of long-term energy homeostasis and food intake.
This theory postulated that the body’s energy stores, in the form of fat, were
somehow affected by the CNS, which regulated energy homeostasis, the balance
between energy consumption, use and storage. The observation that stores
of body fat are almost constant in young animals indicated that long-term
food intake is precisely regulated. Kennedy suggested that, as opposed to a
direct neural pathway that might regulate fat deposition, circulating metabolites were the most likely candidates to regulate long-term food intake. In
contrast to the short-term regulation of food intake by factors such as blood
glucose, insulin and gut fill, the lipostat was postulated as a system that
modulates a slower response to deficits in whole-animal energy reserves.
Thus, when energy reserves, in the form of adipose tissue, were high, the
satiety centres in the hypothalamus were activated and food intake was
reduced. During fasting and starvation, adipose reserves are mobilized for
energy, and appetite increases concurrently.
Evidence for the lipostat theory was provided by several studies using
the technique of parabiosis. In this method, the circulatory system of two
animals is surgically connected. This allows one to observe the blood-borne
effects of experimental manipulation of one animal on its conjoined partner.
Hervey (1959) studied the systemic effects of an obese rat on a conjoined
normal rat (Fig. 11.2). To induce obesity, he destroyed the VMH region. This
area of the hypothalamus inhibits food intake. Its destruction induced
hyperphagia (excessive eating) and, subsequently, obesity. He then surgically joined these obese, hyperphagic rats in parabiosis with normal rats.
The normal rats stopped eating and lost weight. This provided strong evidence that some factor in the circulation (a ‘satiety signal’) from the obese rat
was affecting the VMH of the normal rat to reduce food intake. Later studies by other scientists in the 1980s showed that this satiety factor mediated
feeding in other models of obesity. Thus, rats with diet-induced obesity
parabiosed with non-obese rats reduced the food intake of the normal rats.
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Normal rat
Obese rat:
VMH ablation
or diet-induced
Loss of appetite,
weight loss in
normal rats
ob/ob mouse:
hyperphagic
obese,
insulin-resistant
db/db mouse:
hyperphagic
obese,
insulin-resistant
Loss of appetite,
weight loss,
death in ob/ob mice
Fig. 11.2. Effects of parabiosis on feed intake and body weight in rodents.
The body weight loss of the normal rats was due to a selective reduction in
body fat. This was attributed to an ‘energy stabilizing’ factor that regulated
energy homeostasis in the body that was carried in the circulation from the
obese to the normal rat. These elegant whole-animal experiments led to
the conclusion that regulation of appetite (and thus body energy reserves in
the form of fat) was likely mediated by a blood-borne factor, either a hormone
or a metabolite, which acted at the level of the hypothalamus.
To examine this phenomenon further, two widely used animal models
for diabetes and obesity, the diabetic mouse (db/db) and the obese mouse
(ob/ob), were used. It was known that these strains were the result of single
gene mutations, although the identity of the affected genes was unknown.
Thus, these animals provided excellent models with which to examine single gene effects on humoral-mediated food intake and obesity. Both strains
of mice exhibit the characteristics of Type II (insulin-resistant) diabetes and
are obese, hyperphagic, hyperglycaemic and hyperinsulinaemic. When the
db/db and ob/ob mice were joined in parabiosis, the ob/ob mice became
anorexic, hypoglycaemic and died of starvation. This suggested that the two
strains of mice had defects in different genes, which resulted in a similar
phenotype. In addition, the results suggested that the db/db mice produced
a blood-borne factor that was absent in the ob/ob mice which inhibits food
intake. Thus, the ob/ob mice react to a ‘satiety signal’, which is produced by
the db/db mice but is ineffective in the db/db mice. It is now known that the
ob/ob mice are leptin-deficient and the db/db mice produce leptin but have
no functional leptin receptors. These observations set the stage for the discovery of leptin, a blood-borne satiety factor that regulates long-term energy
homeostasis (Table 11.1).
Leptin, Body Composition and Appetite Control
207
Table 11.1. Factors that regulate food intake.
Short-term signals
Short-term responses
A. Sensory inputs: sight, smell,
taste, texture, memory
Size, duration, frequency of meals
B. Nutrient inputs: glucose, fatty acids,
Ingestion, digestion, absorption of nutrients
amino acids
C. Gut signals: chemoreceptors,
mechanoreceptors, gut hormones
Food-induced thermogenesis
Long-term signals
Long-term responses
Circulating nutrients, hormones
and metabolites
Hunger/satiety
Glycogen, lipid energy storage
Thermogenesis, energy expenditure
The Discovery of Leptin
Dr Friedman’s group at Rockefeller University used the ob/ob mouse and
sophisticated molecular biology techniques to search for and eventually isolate the product of the obese gene. In 1994, Friedman and his colleagues
reported the positional cloning and characterization of the obese gene from
white adipose tissue of the ob/ob mice. Friedman showed that in the ob/ob
mice this gene contained a mutation that interrupts the transcription of the
ob gene product, resulting in a biologically inactive molecule. The absence of
the product of this gene in ob/ob mice was responsible for overeating and
obesity in these mice. The natural product of this gene in wild-type mice was
dubbed leptin (from the Greek word leptos, meaning thin). Leptin treatment
of the ob/ob mice reduced feed intake and lowered body weights. The reduction in body weight occurred specifically through a selective loss of fat mass.
The circulating levels of this hormone were highly correlated with fat mass.
Thus, leptin fulfilled the role of the long-sought ‘lipostat’, proposed by
Kennedy, a humoral factor that was proportional to fat mass and regulated
energy homeostasis and food intake (Fig. 11.3). Not unimportantly, this was
also the first indication that adipose tissue acted not only as a passive energy
storage depot, but also as an endocrine organ, secreting a satiety factor that
regulated food intake and energy homeostasis. Leptin is believed to be an
important regulator of appetite, energy metabolism and body composition,
essential factors in efficient farm animal production.
The leptin gene and molecule
The ob gene, which codes for leptin, consists of 4500 bp. A nonsense mutation at position 105 of the gene interrupts the transcription of the ob gene in
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Chapter 11
Hypothalamus
(-)
Food intake
Energy expenditure
Leptin
Adipose tissue
Autocrine
loop
Fig. 11.3. The production and effects of leptin.
ob/ob mice and produces a truncated, biologically inactive molecule in these
animals. The leptin molecule is a single-chain polypeptide containing 146
amino acids and circulates as a 16,000 Da peptide. The molecule is highly
conserved and displays an 84% amino acid sequence identity between mice
and humans. Leptin is synthesized and secreted primarily by white adipose
tissue. The leptin gene has been cloned from several domestic animal
species, including pigs, cattle, sheep and chickens.
Leptin receptors and binding proteins
The receptors for leptin are members of the Class I cytokine receptor family.
They consist of a single protein chain that traverses the plasma membrane
(Fig. 11.4). Ligand binding induces dimerization and activation of the receptor. Five isoforms of the leptin receptor exist, composed of 830 to 1162 amino
acids, and are designated Ob-Ra through Ob-Re. The isoforms are derived
from different splicing of a single gene and differ from one another in their
overall length and the extent of their cytoplasmic domain. Only one of these
receptors, Ob-Rb, contains all domains, extracellular, transmembrane and
intracellular, needed for a fully active receptor. The Ob-Rb receptor is
believed to mediate leptin’s effects on appetite and food intake. This receptor contains the Box 1 and Box 2 motifs that are important for signal transduction via the JAK/STAT pathway. Box 1 serves as a binding site for JAK2,
while Box 2 binds STAT3. The Ob-Rb receptor is found in low levels in many
tissues of the body, but is especially abundant in specific nuclei of the hypothalamus that are involved in feeding and satiety. The ARC nucleus, the
VMH and the paraventricular nucleus (PVN) all contain the Ob-Rb receptor,
Leptin, Body Composition and Appetite Control
Plasma
membrane
209
Box 1
Ob-Re
830
amino acids
Box 2
Box 3
Ob-Ra
Ob-Rc
Ob-Rd
Ob-Rb
892-900
amino acids
1632
amino acids
Fig. 11.4. Molecular forms of the leptin receptors.
marking these feeding and satiety centres as the hypothalamic targets of leptin. Many of the CNS-mediated effects of leptin are mediated by interaction
with the ARC nucleus. The ARC nucleus is the site of synthesis of the neurotransmitter, neuropeptide Y (NPY). NPY is potent inducer of appetite and
food intake. The primary effect of leptin on the inhibition of feeding is
believed to be via the inhibition of the synthesis and release of NPY by the
ARC. The Ob-Rb receptor is also found in the LH, a region considered to be
involved in feeding, not satiety. Finally, the Ob-Rb receptor is found in the
dorsomedial nucleus (DMN) of the hypothalamus. This region contains neurones that have complex autonomic effects on long-term growth and body
composition. More recent evidence suggests that the appetite-suppressing
effects of leptin may involve hypothalamic αMSH, agouti-related protein
(AGRP) and melanin-concentrating hormone (MCH). These hypothalamic
neurotransmitters are discussed at the end of this chapter.
The truncated receptor forms, Ob-Ra, Ob-Rc and Ob-Rd, have a shortened intracellular domain, consisting of 32 to 40 amino acids. These isoforms
are found in many tissues, including skeletal muscle, adipose, kidney, liver
and the choroid plexus but a role for these short isoforms in regulating
appetite has not been demonstrated. The Ob-Ra receptor is abundant in the
choroid plexus of the brain and may act as a transport protein for leptin,
allowing entry into the CNS, bypassing the blood–brain barrier. This truncated receptor also exists in the kidney, where it is thought to play a role in
the clearance of leptin. Leptin has many direct effects on peripheral tissues.
Whether these effects are mediated by the truncated receptors or by the
small amounts of Ob-Rb present in the tissues is unclear. The last short
receptor form, Ob-Re, is a soluble receptor, lacking the transmembrane and
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Chapter 11
cytoplasmic domains. It consists of only a 830-amino acid, extracellular, ligand-binding domain. This receptor is found in the circulation, where it acts
as a binding protein for leptin. In addition, Ob-Re is found in the placenta
and increases to 40-fold during pregnancy, leading the hypothesis that it is
involved in regulating fetal growth and energy use.
Mutations in the leptin receptor that result in a reduction of leptin receptors or in non-functional receptors have been shown to be responsible for obesity in laboratory animals. Just as the absence of leptin itself results in
overeating, obesity and diabetes, animals with defects in leptin receptors
display a similar phenotype. Although circulating leptin concentrations are
elevated, these animals are unable to respond to leptin and are leptin-resistant. For example, the genetic diabetic mice (db/db), discussed above, have
only the truncated Ob-Ra form of the leptin receptor. The obese strain of rats,
Zucker fatty rats (fa/fa), have low numbers of leptin receptors due to a single
amino acid substitution in the receptor molecule.
Like the IGFs, circulating leptin is complexed to serum-binding proteins
in a specific, saturable and reversible manner. Rodents have three circulating
binding proteins with molecular masses of 85, 176 and 240 kDa, while humans
have two binding proteins with masses of 176 and 240 kDa. It appears that leptin binding to these proteins is regulated by physiological processes. Obesity
reduces the proportion of bound leptin, providing more unbound, presumably active leptin, while fasting increases protein-bound leptin binding.
Biological actions of leptin
The biological effects of the leptin have been examined in the obese diabetic
mouse models, ob/ob, db/db and wild-type mice. When injected into the
leptin-deficient ob/ob mice, leptin rapidly (4 days) reduced food intake to
about 40% of the untreated ob/ob mice and reduced their body weight by
about 40% after 33 days of treatment. Pair-fed ob/ob mice, matched by age,
sex and weight, lost less weight than animals treated with leptin. Leptin
treatment of db/db mice did not affect food intake or body weight and later
studies of db/db mice showed them to lack the leptin receptor. Treatment of
wild-type mice with leptin resulted in a small, but significant loss in body
weight of approximately 12% and food intake of about 10%. As might be
expected, these effects required higher doses of leptin than those that were
effective in the leptin-deficient ob/ob mice. Body weight loss in the leptintreated ob/ob mice was due to an almost exclusive loss of adipose tissue
mass. The lean body masses of control and leptin-treated mice were not different from one another. In wild-type mice body fat was reduced from
12.2% to 0.67% by leptin treatment. These early studies demonstrated the
effectiveness of leptin in reducing food intake, body weight and, specifically, the fat mass of leptin-deficient and normal mice. The effects of leptin
on appetite that are mediated via the hypothalamus are indirect, and act
through a reduction of hypothalamic neurotransmitter NPY. Leptin effects
that are mediated by NPY include a reduction of food intake, body weight
Leptin, Body Composition and Appetite Control
211
and fat mass in normal and leptin-deficient animals, induction of puberty
in mice and increased thermogenesis.
In light of the effects of leptin on body fat and appetite, the effects of leptin on energy metabolism were examined. When injected into ob/ob mice,
leptin induced increased oxygen consumption, locomotor activity and body
temperature of these obese mice to values seen in wild-type mice. In addition, the elevated serum insulin and glucose values in these animals were
decreased to near-normal values in a dose-dependent manner by leptin
treatment. None of these parameters was affected by leptin treatment of
wild-type mice. Thus, leptin administration reversed the physiological problems associated with the obese phenotype, including endocrine and energy
homeostasis parameters.
In addition, injection of small amounts of leptin (60 pmol) directly into
the third ventricle of the brain, where it has direct access to the hypothalamus, induced a rapid (within 30 min) reduction of food intake and increased
oxygen consumption in lean and ob/ob mice. These very rapid-onset effects
of leptin on oxygen consumption indicated that the sympathetic nervous
system (SNS) may be involved, and the effects of norepinephrine on brown
adipose tissue metabolism may mediate these effects of leptin.
The effects of leptin on thermogenesis and oxygen consumption may be
mediated via the hypothalamus and sympathetic innervation or may be a
result of direct effects of leptin on peripheral tissues. The thermogenic effects
of leptin are likely mediated by mitochondrial UCPs. The UCPs uncouple
oxidative phosphorylation from ATP generation and increase proton leakage
through the inner mitochondrial membrane. Instead of using the proton
energy of the cytochrome chain for oxidative phosphorylation, the energy is
released as heat, resulting in increased thermogenesis. Different UCPs have
been found in various tissues. Brown adipose tissue contains UCP-1, while
homologous proteins called UCP-2 and UCP-3 have been reported. Several
mammalian tissues containing UCP-2 and UCP-3 are expressed primarily in
skeletal muscle. Leptin directly induces expression of UCP-2 in pancreatic
islets and in epididymal, retroperitoneal and subcutaneous fat of normal
rats. Leptin administered intracerebroventricularly (ICV) also stimulated
UCP mRNA by 70% in rat brown adipose tissue. Leptin treatment of ob/ob
mice stimulated UCP-3 expression in skeletal muscle. Thus, leptin has
important effects on energy metabolism in animals, increasing oxygen consumption and motor activity, increasing thermogenesis by direct induction
of mitochondrial UCPs and by indirect actions through the CNS and the
SNS.
In all studies to date, leptin is more effective in the leptin-deficient ob/ob
animals than in normal animals. In addition, even the highest doses of leptin used did not induce a ‘supranormal’ response. That is, the animals’ metabolic indices, insulin and glucose levels did not exceed normal levels and the
values of wild-type mice were largely unaffected. Most obesity in humans is
diet-induced, and as a result, obese humans have elevated leptin, proportional to their fat mass. As a result, most obese humans have plenty of leptin
and are actually leptin-resistant.
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Chapter 11
Effects of leptin on reproduction
A long held tenet of reproductive biology is that adequate energy stores, in
the form of adipose tissue, are required for efficient reproduction. Adequate
energy reserves are needed to support the growth and development of the
embryo and fetus, parturition and lactation. It is axiomatic in farm animal
production systems that lean females (poor body condition) reproduce less
efficiently than those with moderate body fat. In human athletes, lean
women have a delayed onset of puberty and the reproductive cycles of lean
females are interrupted. As leptin levels are proportional to body fat, it was
hypothesized that leptin may act as the signal to the reproductive system
that sufficient body fat exists to support a successful conception and pregnancy. Leptin treatment of normal female mice induces early puberty, reproductive system maturation and mating. Leptin does this by inducing release
of the reproductive hormones LH and FSH, acting directly on the anterior
pituitary and indirectly, inducing LHRH release by the hypothalamus.
Leptin has similar effects in the food-restricted, infertile ob/ob male mouse,
inducing testicular maturation and normal fertility. Embryonic and fetal leptin concentrations increase during development and are proportional to fetal
size and birth weight. Leptin and leptin receptor mRNA is present in human
placental tissue. These observations suggest that leptin may be involved in
placental–uterine–fetal growth and development and may act locally or in a
hormonal fashion to signal nutrient availability and metabolic regulation of
the fetoplacental unit during embryonic development.
Direct peripheral effects of leptin
In addition to those effects of leptin that are mediated by the hypothalamus, leptin has several direct effects on a variety of tissues. Direct
effects of a hormone or growth factor are difficult to establish using animal models injected with the factor of choice, in vivo. Even if carefully
controlled, whole animal studies are generally ambiguous, due to the
multiple interactions of the exogenous drug with the myriad of regulatory systems in the living animal. For this reason, studies using cells
in vitro are performed to negate extraneous factors under defined conditions. For example, leptin can act as a mitogen, stimulating the proliferation of mouse embryonic fibroblast cells, in vitro. Leptin has direct effects
on white adipose tissue, where it acts through the Ob-Rb receptor. Leptin
suppresses the expression of adipocyte ACC, an enzyme required for
lipogenesis, while stimulating lipolysis. In C2C12 cells, leptin stimulates
glucose transport and glycogen synthesis at physiological levels. Leptin
also stimulates pancreatic islet UCP-2 mRNA, mRNA for enzymes
involved with free fatty acid oxidation, and inhibits fatty acid esterification. In the reproductive system, leptin has direct effects on inducing the
release of FSH and LH from anterior pituitaries and LHRH release from
the hypothalamus in vitro.
Leptin, Body Composition and Appetite Control
213
Recent studies have shown that both long and short forms of the leptin
receptors are present in haematopoietic organs, endothelial cells and keratinocytes. In haematopoietic cells, leptin induces cell survival, proliferation
and differentiation. In endothelial cells, leptin stimulates the formation of
new blood vessels (angiogenesis) and the local expression of leptin and its
receptors in the ovary, uterine endometrium and placenta has led to suggestions that leptin may be involved in the extensive vascularization/devascularization that occurs in these tissues during the reproductive cycle and
pregnancy. Leptin is also involved in stimulating the proliferation of keratinocytes during skin repair and is thought to be involved in wound healing. There are high levels of leptin and the leptin receptor in fetal bone and
cartilage. Leptin, acting via the CNS, and probably locally, inhibits bone
formation and ob/ob mice have high bone masses.
The direct effects of leptin on many different tissues suggest that this
hormone is involved in the coordination of many physiological processes.
Leptin acts not only on the hypothalamus to regulate food intake, but also
acts as a peripheral hormone and as a local autocrine and paracrine growth
factor in its own right.
Regulation of leptin concentrations
Circulating concentrations of leptin are modulated by several physiological
states. As we have seen, the concentration of circulating leptin is proportional to fat mass. Leptin mRNA concentrations are higher in subcutaneous
fat than in omental fat. Leptin concentrations are reduced by fasting and by
the antidiabetic drugs, the TZDs. Leptin concentrations are sexually dimorphic, and are increased in females, independent of adiposity. When fat mass
is accounted for, females have about twice the amount of circulating leptin
than males. Leptin concentrations vary diurnally and are higher at night
than during the daytime. The hormones insulin, glucocorticoids and GH
induce an increase in leptin. As might be expected nutritional status affects
leptin concentrations, and a chronic high fat diet increases circulating levels
of leptin. Leptin is elevated during pregnancy. Testosterone and chronic GH
lower leptin levels. The physiological conditions of exercise, cold exposure
and cold-induced feed intake have no effect on leptin concentrations.
Significance of leptin to animal production
One may wonder about the significance to animal production of a hormone
that reduces appetite, body weight and feed intake. The discovery of leptin
provided the first link between the systemic circulation and the CNS that
regulated long-term energy balance. As the CNS is a protected site, entrance
into this sanctuary is highly regulated and limited to a select few. This
ensures the protection of the CNS from the myriad of toxins, metabolites and
environmental chemical challenges encountered on a daily basis. A naturally
214
Chapter 11
occurring circulating hormone that can pass through the blood–brain barrier
and alter feeding behaviour is of immense importance.
Leptin’s effects on animal energy metabolism are not in keeping with
efficient animal production. As leptin increases oxygen consumption, and
thus thermogenesis and metabolic rate, by uncoupling oxidative phosphorylation, the use of leptin or leptin agonists for animal production is counterintuitive. To make use of leptin’s potential effects on energy metabolism, the
use of leptin antagonists, which block the effects of leptin, would be beneficial. A leptin mutant, consisting of a single amino acid alteration, has been
described. This antagonist induces weight gain in mice and may provide the
advantages of quicker growth and earlier fat deposition in economically
important animals. Studies to identify small molecules that are orally active,
cross the blood–brain barrier and antagonize the effects of leptin are being
explored for use in production animals.
The effects of leptin on reproductive system processes are striking. The
communication of adipose tissue with the reproductive system via leptin is
unique and complete as it involves all portions of the reproductive system:
hypothalamus, pituitary, ovaries and testes. In addition, leptin may be
involved in regulation of fetal, placental and uterine metabolism and angiogenesis. Leptin’s broad effects on reproduction and embryonic development
endow it with a true reproductive hormone status. Leptin may be useful to
induce early puberty in slightly thinner, younger animals and may be used
to shorten the interval from parturition to oestrus. These effects, as well as
possible role in fetal growth and development, may enhance the reproductive efficiency of farm animals.
Appetite-inducing (Orexigenic) Peptides of the Hypothalamus
Appetite is highly regulated in vertebrates and chemically coded in the
hypothalamus. The hypothalamus consists of a neural network of appetiteinducing (orexigenic) and appetite-inhibiting (anorexigenic) signals which
are modulated by nutrients, neural stimuli and hormones. Some of the more
important hypothalamic regulators of appetite are outlined.
NPY is a major regulator of appetite and plays a role in many appetiteregulating mechanisms. NPY is thought to be the naturally occurring
appetite transducer and the essential component of the final pathway in the
hypothalamic integration of energy homeostasis. It is the only messenger
molecule that can be considered as a physiological appetite transducer in the
brain. NPY is present in the ARC and dorsomedial nuclei. Its amino acid
sequence is related to pancreatic polypeptide. NPY is potent appetite stimulant, which is released in the dark in nocturnal feeding rodents and in anticipation of scheduled feeding. There is increased release during fasting. In the
obese, leptin-resistant rodent models, ob/ob mice, fa/fa rats and db/db mice,
an increase in hypothalamic NPY expression accompanies the obese, hyperphagic phenotype of these animals. NPY expression in these animals is
Leptin, Body Composition and Appetite Control
215
reduced by leptin treatment. In physiological states that require increased
energy resources, such as lactation, hypothalamic expression of NPY is
increased, leading to increased appetite and food consumption. This provides the animal with the energy necessary to support the increased energy
demand for milk synthesis.
Galanin (gal) is another peptide (29 amino acids) found throughout the
hypothalamus. As NPY neurones may induce gal synthesis, gal, through βendorphins, may mediate the effects of NPY, acting to induce epinephrine
release. As there is no correlation of gal expression with daily feeding patterns and as fasting reduces gal expression, its role in the regulation of daily
feeding is uncertain.
Orexins, also called hypocretins, are produced in many places in the body,
including the LH, the gut, pancreas, kidney and the adrenal and thyroid
glands. The orexins are the product of a single gene which produces two peptides by alternative splicing, orexin A (33 amino acids) and orexin B (28 amino
acids). ICV injection of orexins increases food intake and wakefulness. ORX-A
increases heart rate, blood pressure, intestinal motility and gastric acid secretion, while both orexins induce epinephrine/norepinephrine and insulin
release. This suggests that orexins may play a role in the mediation of the
stress response. The acute, short-term effects of orexins on feeding may be secondary to effects on wakefulness and activity. The orexigenic effects of the
orexins are mediated by an induction of NPY expression. Orexins are induced
by reduced blood glucose levels, while increased circulating levels of glucose
and leptin reduce orexin expression. There are two receptors for the orexins.
Orexin receptor mutations induce narcolepsy in dogs and humans.
Other hypothalamic peptides which are orexigenic include opioid peptides (e.g. β-endorphin), which have a short-lived modest effect, and MCH
which may potentiate nocturnal feeding. Anorexic signals in the hypothalamus, which balance and antagonize orexic signals, include CRH, considered a tonic restraint of appetite, and urocortin (a CRH-related peptide),
which is more potent than CRH. In addition, neurotensin, glucagon-like
peptide-1, melanocortin and AGRP are potential anorexic peptides.
References and Further Reading
Auwerx, J. and Staels, B. (1998) Leptin. The
Lancet 351, 737–742.
Conway, G.S. and Jacobs, H.S. (1997) Leptin:
a hormone of reproduction. Human
Reproduction 12, 633–635.
Flier, J.S. (1997) Leptin expression and action:
new
experimental
paradigms.
Proceedings of the National Academy of
Sciences USA 94, 4242–4245.
Flier, J.S. and Lowell, B.B. (1997) Obesity
research springs a protein leak. Nature
Genetics 15, 223–224.
Flier, J.S. and Maratos-Flier, E. (1998)
Obesity and the hypothalamus: novel
peptides for new pathways. Cell 92,
427–440.
Frisch, R.E. and Revelle, R. (1970) Height and
weight at menarche and a hypothesis of
critical body weights and adolescent
events. Science 169, 397–399.
Forbes, J.M. (1995) Voluntary Food Intake and
Diet Selection in Farm Animals.
CAB International, Wallingford, UK,
532 pp.
216
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Friedman, J.M. (2002) The function of leptin
in nutrition, weight and physiology.
Nutrition Reviews 60, S1–S14.
Fruhbeck, G. (2002) Peripheral actions of leptin and its involvement in disease.
Nutrition Reviews 60, S47–S55.
Harrold, J.A. (2004) Leptin leads hypothalamic feeding circuits in a new direction.
Bioessays 26, 1043–1045.
Hervey, G.R. (1959) The effects of lesions in
the hypothalamus in parabiotic rats.
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336–352.
Hossner, K.L. (1998) Cellular, molecular and
physiological aspects of leptin: potential
application in animal production. Canadian
Journal of Animal Science 78, 463–472.
Kennedy, G.C. (1953) The role of depot fat in
the hypothalamic control of food intake
in the rat. Proceedings of the Royal Society
of London B140, 578–592.
Tartaglia, L.A. (1997) The leptin receptor.
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6093–6096.
Wilding, J.P.H. (2002) Neuropeptides and
appetite control. Diabetic Medicine 19,
619–627.
Zhang, Y., Proenca, R., Maffei, M., Barone, M.,
Leopold, L. and Friedman, J.M. (1994)
Positional cloning of the mouse obese
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372, 425–431.
Index
3T3-F442A cells 164, 174, 175
3T3-L1 cells 164–165, 166, 168, 170, 171, 173,
174, 175, 176, 177
5α-reductase 183
αGPDH 78
ACC 197, 212
acetycholine 76, 193
acetyl-CoA 88, 89, 92, 167
acetyl-coenzyme A carboxylase (ACC)
84, 88, 104, 168, 174
acid phosphatase 65, 131, 133, 134
acromegaly 104
ACTH 176
actin 68, 71, 73, 74, 76, 77, 79
activin 140, 141
acyl-CoA 91
ADD1/SREBP1 167, 169, 170–172, 174
adenylate cyclase 50, 130, 196
adipoblast 84, 86
adipocyte 163, 164
adipogenesis 84, 167, 168, 169, 170, 171,
173, 176
adiponectin 178
adipose tissue 82
adipsin 166, 176
adrenal cortex 41
adrenal gland 41, 181, 191
adrenal medulla 41, 193, 194, 195
adrenaline see epinephrine
adrenergic receptors 195–198
adrenocorticosteroids see glucocorticoids
AGRP 209, 215
alkaline phosphatase 63, 136, 139, 142
allophenic mice 80
ampicillin 31
androgens 182, 183, 186–187, 188–189
angiotensin 179
angiotensinogen 179
anticodon 22
ap2 166, 168, 169
appetite 202, 204, 209, 210, 214
arachidonic acid 168, 176
arcuate (ARC) nucleus 96, 204, 208,
209, 214
ATP 63, 196
autocrine 37
autonomic nervous system (ANS) 192, 193
average daily gain 7
β-adrenergic receptors 196–198, 199, 200
β-agonists 42, 158, 196, 198, 199, 200
β-endorphins 215
beta oxidation 91–92, 167, 168
bHLH transcription factor 148, 149, 171
biglycan 62
biotechnology 27
blastocyst 55
body weight 2–3
bone 60, 61
bone canaliculi 64
bone lacuni 64
bone marrow 60, 64, 67
bone marrow stroma 133, 134, 142
bone matrix 61, 62, 65, 67
bone mineralization see ossification
217
218
Index
bone morphogenic protein (BMP) 133, 135,
136, 137, 140, 141, 143–144,
155–156, 157
Booroola gene 28
brown adipose 82, 83, 163, 164, 170, 173,
198, 211
C/EBP 167, 169–170, 173, 176, 177
C/EBP knockouts 170
C2C12 cells 148, 153, 212
C3H10T1/2 164, 167
calbindin 128
calcitonin 40, 124, 129–130, 135
calcium 76, 123, 124, 125, 128, 129, 130, 131
calcium phosphate 63
calcium pump 129
Callipyge gene 6, 79
calpains 79, 80, 200
calpastatins 79, 80, 200
cAMP 50, 51, 90, 96, 97, 121, 130, 165, 176,
177, 196, 197
cAMP response element binding protein
(CREBP) 121, 196
cancellous bone 61, 69
carbonic anhydrase 65, 131
carnitine 92
catecholamines 41, 42, 193, 194, 195
cathepsin K 65
cathepsins 79, 200
Cbfa1 133
CCAAT elements 169
C-cells see parafollicular cells
Cdks 17
cell cycle 16–17
cell differentiation 59
cell proliferation 59
Central Dogma of Cell Biology 18
chimera 34, 80
cholecalciferol 127
chondroblast 61
chondrocyte 61, 67, 131, 142, 143, 144
chondroitin sulphate 62, 63
choroid plexus 209
chromatin 13
chromosomes, eukaryote 19
chylomicrons 87
cimaterol 198
citric acid cycle 92
clenbuterol 198, 200
c-Met 152, 157, 158
CNS 191, 192, 193, 195, 204, 205, 211, 213
codons 22
cold stress 10–11
collagen 62, 63, 133, 134, 139, 142, 157
collagenase 62, 65, 133, 137, 139, 142
complementary DNA (cDNA) 29
corpus luteum 182
corticosteroids see glucocorticoids
cortisol 189
creatine kinase 148
Cre-lox recombinase 36
critical period theory 86
CSF-I 143
Cushing’s disease 176
cyclins 17
cytokine/haematopoietin receptor 99
db/db mouse 206, 210, 214
decorin 62, 157
dermatin sulphate 62
dermomyotome 57
desmin 71, 79
diabetes 159, 167, 206
diacylglycerol (DAG) 51, 53
diaphysis 61
dienestrol 181
diethylstilbestrol (DES) 181
dihydrotestosterone (DHT) 183, 188
DNA replication 18
DNA sticky ends 30
dopamine 45, 96, 194, 195
dorsomedial nucleus (DMN) 209
double-muscling 6, 156
DR-1 site 167
DRIP/TRAP/ARC complex 173
dual effector hypothesis 115–116
dwarfism 104–105
E-boxes 149, 171
E proteins 149
ectoderm 57
EGF 140, 177
elastase 142
embryogenesis 55
embryonic stem cells 34
endochondral bone see cancellous bone
endoderm 57
endoplasmic reticulum 15, 62
endosteum 61
epaxial muscles 69
epinephrine 41, 88, 90, 191, 194, 195, 196,
197, 215
epiphyseal plate 61, 67, 183
epiphysis 61
eukaryotes 13
eukaryotic organelles 14–16
exons 19
exteroceptive effects 45, 47
Index
extracellular matrix (ECM) 57, 58, 60, 63, 64,
67, 111, 132, 135, 136, 139, 140, 141,
142, 143, 147, 155, 157, 158, 159, 177
fa/fa rat 210, 214
fat depots 83–84, 86
fatty acid catabolism 91–92
fatty acid synthase (FAS) 84, 88, 104, 166,
168, 171, 174, 175
fatty acid transporter 166
fatty acids 87, 89, 90, 91, 96, 104
feedback control 44
FGF 133, 135–137, 142, 152, 156, 177
fibroblast 57-58, 61, 84, 142, 170, 171
fibronectin 62, 63, 157
fight or flight 191
food intake 202, 204, 210, 215
food preference 202
GABA 96
galanin 215
gastrulation 56–57, 69
gelatinase 65, 139
gene isolation 29
gene promoter 24
gene transcription 18, 20, 21, 24–26
genes, prokaryote 19
genetic markers 7–8
genetics 6
genomic library 29
germ layers 56
GH 94, 95, 96–98, 112, 113, 114, 116, 117, 155,
187, 188, 189
adipose 174–175
carbohydrates 102–103
growth 115, 117, 118
insulin 103–104
lactation 117
leptin 213
lipids 103–104, 175
metabolic effects 101–103
muscle 153, 159, 160
proteins 102
receptor 49, 99–101
selection marker 8
species specificity 102
steroids 117
target organs 102
transgenics 119–121
GH secretagogue receptor (GHS) 98
GHBP 100–101
ghrelin 98–99
GHRH 44, 96, 98, 99, 105, 188
GHRIH see somatostatin
219
gigantism 104
glucagon 42, 88, 90, 195
glucocorticoids 133–134, 158, 160, 165, 174,
175, 176, 178, 181, 182, 189, 213
glucostat theory 204
GLUT4 104
glycerol 87, 90, 104
glycolysis 77, 78
glycosaminoglycans 63
GnRH 45
Golgi apparatus 15, 62, 64
gonads 42
GPDH 166, 175, 176
GPLR 48, 49, 51
G-proteins 49–50
growth 2, 3–4
growth and differentiation factor (GDF)
142, 143
growth curves 4–6
growth plate see epiphyseal plate
gut fill 205
heat stress 11–12
hedgehogs 131
heparin sulphate 156, 158
hepatocyte growth factor (HGF)
137, 152, 157–158
heterologous gene recombination 33
hexestrol 182
histone 13, 19
histone acetyl transferase (HAT) 173
histone deacetylase 173
homologous gene recombination 33–34
hormone definition 37
hormone receptors 46
hormone types 42–43
hormones and genetic selection 8
hormone-sensitive lipase 90, 196
hyaluronic acid 63
hydroxyapatite 63, 65, 123
hydroxyproline 131
hypaxial muscles 69
hyperphagia 205, 206
hyperplasia 2, 86
hypertrophy 2, 6, 86
hypocretins see orexins
hypophyseal portal system 44
hypothalamic nuclei 43, 44
hypothalamus 43, 44, 45, 204, 205, 206, 208,
211, 212, 214
IBMX 165, 177
IDH 80, 82
220
Index
IGF 94, 96, 178, 188, 189
bone 132, 133, 134, 135, 137, 139,
153–155
domains 107
genes 107
growth effects 112–113
knockouts 114, 154
metabolic effects 111–112
muscle 153–155, 157, 159, 160
receptors 108–109
selection marker 8
structure 107
IGF-I receptor 49
IGF-II receptor 108–109
IGFBP 110–111, 132, 139, 175
inner cell mass 55
insulin 42, 96, 108, 109, 112, 120, 121, 158–159,
165, 171, 173–174, 175, 176, 178, 179,
195, 204, 205, 211, 213, 215
insulin receptor 49
integrin 111
integrin receptor 62, 63, 65
interleukins 139
intermediate filaments 71
interphase 16
intron 14, 19–20
IP3 51, 53
IRS-1 49, 176
isoproterenol 196, 198
isozymes 80, 82
Janus kinase (JAK) 48, 49, 100, 208
keratin sulfate 63
kinases 17
knockout mice 34–36
Lbx1 152, 157
leptin 83, 99, 166, 168, 169, 178, 204, 206, 207,
208, 209, 210, 211, 212, 213, 215
antagonist 214
binding proteins 210
direct effects 212–213
receptors 208–210, 212, 213
limb bud 69, 131, 136, 138, 147, 149, 156–157
lipogenesis 87
lipolysis 87, 104
lipoprotein lipase (LPL) 84, 87, 166, 168, 171,
174, 175, 176
lipoproteins 87
lipostat theory 205, 207
lysosomes 15, 64
M-6-P receptor 108–109
macrophage 84
malonyl-CoA 88, 89
MAP kinase 154, 169
maximum fat cell size theory 86
MCH 209, 215
median eminence 96
melengestrol acetate (MGA) 182
membranous bone 61
mesenchyme 57, 61, 84, 131, 137, 138, 142, 144
mesoderm 57, 61
metallothionein 119, 120
metaphysis 61
mitochondria 15–16, 62, 64
mitogen-activated protein (MAP) kinase 49
mitosis 16–17
monocytes 64
morphogens 131
motor neurones 75
MRF4 149, 150–151, 154
mRNA 21–22, 29
MSA 106, 107
MSH 209
multiple locus markers 9
muscle fibre see myofibre
muscle fibre types 77–78
muscle organelles 74–75
muscle regulatory factors (MRF) 148–152,
155, 157, 171
muscle synapse 75
Myf 5 149, 150–152, 154
myoblast 57, 68, 69, 71, 146, 148, 149, 150,
151, 153, 154, 155, 157, 158, 170, 212
MyoD 148, 149, 150, 151, 152, 154, 155, 156,
157, 159
myofibre 68, 69, 70, 74, 77, 146, 148, 150, 151,
153, 154, 155, 156, 157, 200
myofibrillar proteins 68, 69, 71
myogenesis 146, 147, 157
myogenic cell systems 147–148
myogenin 148, 149, 150, 151, 154, 155, 156,
157, 159
myoglobin 77
myosin 68, 71, 73, 74, 76, 77, 79, 148
myosin ATPase 74, 76, 78
myosin heavy chain (MHC) 73, 78
myostatin 6, 155
myotubes 68, 69, 71, 146, 148, 150, 154, 155,
157, 158
NADPH 89
neomycin 34
neural tube 147
neurohormone 37, 43, 44, 45
neurohypophysis see posterior pituitary
Index
neuropeptide Y (NPY) 99, 209, 210, 214, 215
neurotension 215
neurotransmitters 38, 96
NIH 3T3 cells 164
nonshivering-thermogenesis 83
noradrenaline see norepinephrine
norepinephrine 41, 88, 90, 191, 193, 194, 195,
196, 197, 211, 215
notch 131
NSILA 106
nucleus 15
nutrients 9
OB 1771 cell line 176
ob/ob mouse 206, 207, 208, 210, 211, 212, 214
obese gene 207
obesity 204, 205, 206, 210, 211
oestradiol benzoate 181
oestradiol-17β 181, 185, 186, 187, 188, 189
oestrogen 42, 181, 183, 184, 187, 188, 189
oestrogen, bone 134, 135, 137
oestrogen receptor 187
oestrus 183
oleic acid 89
Optaflexx® 198
orexins 215
organic matrix 63, 67
ossification 60, 61, 63
osteoblasts 60, 61, 62, 63, 65, 66, 67, 123, 125,
126, 129, 132, 133, 134, 135, 136, 137,
139, 140, 142, 143, 144
osteocalcin 63, 129, 136, 139, 142
osteochondrosis 60
osteoclasts 60, 61, 64, 65, 66, 67, 125, 126, 129,
130, 131, 132, 133, 134, 139, 140, 142,
143, 144
osteocytes 61, 62, 63, 123, 131, 136, 142
osteogenic protein 143
osteoid see organic matrix
osteomalacia 129
osteonectin 63, 129, 139
osteopontin 63, 136
osteoporosis 135
osteoproterogen (OPG) 66, 67
p 160/CBP/p300 complex 173
palindromes 30
palmitic acid 89
pancreas 42, 97
parabiosis 205
paracrine 37
parafollicular cells 129, 130
parasympathetic nervous system 192, 193
paraventricular nucleus (PVN) 97
221
pars distalis 38
pars tuberalis 40
patched (ptc) gene 131
Pax3 152, 157
Paylean® 198, 200
pBR322 31, 32
PDGF 62, 133, 135, 137–140, 142, 177
PDGF receptors 138
PEPCK 120–121, 168, 176
periosteum 61, 64
peroxisomes 15, 167, 168
PGC-1, -2 173
pGEM 32
PGF2α 176
PGI2 (prostacyclin) 176, 179
phagocytes 64
phosphatases 17, 48
phosphate 123, 125, 129
phospholipase-C 50, 51
photoperiod 10, 45
PI-3 kinase 49, 154
PIP2 51, 53
pituitary gland 38–40
PKA 196, 197
placental lactogen 95, 99, 113
plasma membrane 14–15
plasmids 29, 31, 94
plasmin 140
pluripotent 56
posterior pituitary 40, 45
postganglionic neurones 193, 194
PPAR 167–169, 170, 171, 173, 174, 176, 177,
178, 179
preadipocytes 84, 163, 164, 174, 175, 177, 178
preganglionic neurones 193, 194
progesterone 182, 185
progestins 182, 183
proinsulin 107
prokaryotes 13
prolactin 45, 95, 97, 99, 113
prostaglandins 168, 176
protein kinase A (PKA) 50
protein kinase C (PKC) 51
proteins 19, 46
protein translation 18, 20, 22
proteoglycans 62
PTH 124, 125, 129, 133, 134, 135, 137, 139, 142
pyruvate kinase 176
quantitative trait loci 9
quantitative traits 6
ractopamine 198, 200
Ralgro® 185
222
Index
RANK/RANKL system 66–67, 126, 129, 133,
134, 143
Rathke’s pouch 38, 40
receptor domains 46–47
receptor types 46–47, 49
recombinant DNA 27, 28, 29, 31, 32, 33
releasing hormones 43
renin-angiotensin 179
repartitioning agents 198
resistin 179
restriction enzymes 29–30, 31
restriction fragment length polymorphisms
(RFLP) 8
retinoic acid 177
Revalor-S 189
RGD sequence 62, 63, 65, 111
ribosomes 22
rickets 129
RNA polymerases 19–20, 21, 23–24
RNA types 21, 22
RXR 167, 169, 177
sarcolemma 75, 76
sarcomeres 71, 72, 76, 77
sarcoplasmic reticulum 76
satellite cells 69, 70, 71, 153, 154, 155, 156,
157, 158
satellite DNA 9
satiety factor 205, 206, 207
saturated fats 90
scatter factor see HGF
sclerotome 57
SDH 78
second messengers 47, 48–49, 54
sella turcica 38
serine/threonine kinases 141, 143
sex steroids see oestrogen, testosterone
SHH 151, 152
signal peptide 15
Sirt1 173
Smads 141, 143
smoothened (smo) gene 131
somatic nervous system 193
somatomedin see IGF
somatomedin hypothesis 106–107,
115, 116
somatostatin 45, 97, 98
somatotropes 94
somatotropin see growth hormone
somites 57, 68, 147, 149, 150, 151, 152, 156,
157, 158
splanchnomegaly 104
STATs 48, 49, 208
stearic acid 89
stearoyl-CoA desaturase (SCD) 89, 168, 169
steroid receptors 52–53
sterol response elements (SRE) 171
stress 191
stretch receptors
stromovascular cells 84, 87, 163, 164,
174, 175
stunting 9
sulphation factor 105
SWI/SNF 173
sympathetic nervous system (SNS) 191, 193,
194, 195, 196, 211
sympathoadrenal system 193
Synovex-S 185
T4 ligase 32
temperature, environmental 10, 45
testosterone 42, 134, 183, 188, 213
tetracycline 31
TGFα 140, 176, 177
TGFβ 62, 131, 136, 137, 139, 140–143, 156,
157, 177
TGFβ receptor 141, 142
thermostatic theory 204
thiazolidinediones (TZD) 167, 168, 178,
179, 213
thrombospondin 62–63
thyroid gland 40, 130
thyroid hormones 135, 158, 160, 189
thyroxine (T4) 40, 189
tibial dyschondroplasia 60
titin 73, 74, 79
TNF 66, 139, 178
totipotent 56
transcription coactivators 172, 173
transcription co-repressors 172, 173
transcription factors 24, 25, 26–27, 46, 52
basic helix–loop–helix (bHLH)
proteins 27
C/EBP or CAAT/enhancer-binding
protein 27
homeodomain proteins 26
leucine zipper proteins 27
zinc finger proteins 26
transformed cells 140
transgenics 27, 28, 33
trenbolone acetate (TBA) 186, 187,
188, 189
TRH 45, 97, 98
triacyglycerols 87, 90
triiodothyronine 40
trophoblast 55
tropomyosin 74, 76
troponin 74, 76, 79
TSH 97
T-tubules 75–76
Index
223
tyrosine kinase 47, 49, 108, 121, 131, 136,
138, 158
vitamin D 124, 126–127, 128, 129, 135, 139, 142
vitamin D receptor 127, 128
ubiquitin 17
UCP 83, 170, 173, 211, 212
white adipose 83, 84
wingless (wnt) gene 131, 151
ventromedial hypothalamus (VMH) 204,
205, 208
VFA 204
Z-disc 73, 74, 79
zeranol 181
zygote 55
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